Nucleotide sequences encoding mammalian calcium activated chloride channel-adhesion molecules

ABSTRACT

Nucleotide sequences which encode a mammalian lung endothelial cell adhesion molecule are disclosed. Also disclosed are nucleotide sequences which encode a lung endothelial cell adhesion molecule-associated protein. Recombinant lung endothelial cell adhesion molecule or recombinant lung endothelial cell adhesion molecule-associated protein may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant lung endothelial cell adhesion molecule-associated protein or recombinant lung endothelial cell adhesion molecule-associated protein from the culture medium.

This application claims the priority of a Provisional Application Ser.No. 60/065,922 filed on Nov. 17, 1997, which disclosure is hereinincorporated herein by reference.

This invention was made with government support under grants CA 47668and 09682 from the National Cancer Institute. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to nucleotide sequences encoding a familyof mammalian calcium activated chloride channels which mayalternatively, or additionally function as adhesion molecules. Moreparticularly, the invention is directed to genes isolated from bovineendothelial cells, human endothelial cells and murine endothelial cells,which encode calcium activated chloride channel molecules and includethe lung-endothelial cell adhesion molecules (Lu-ECAM-1) and associatedproteins.

BACKGROUND OF THE INVENTION

Calcium Activated Chloride Channels

Ion channels are not only required for normal cellular functions butalso play a critical role in numerous diseased states. For example,cystic fibrosis results when ion transport in epithelial cells ofindividuals is altered due to a genetic defect of the cystic fibrosistransmembrane conductance regulator (CFTR; Knowles et al., 1983, J.Clin. Invest. 71:1410-1417). Although serious airway pathology isusually the primary cause of mortality in young adults with CF,intestinal epithelial alterations have also been observed. However, theseverity of tissue lesions does not correlate with the expression ofCFTR in humans or mice, suggesting the involvement of cell-specificchannels in addition to CFTR. Further support for the involvement ofother channel protein molecules in CF comes from observations thatcalcium activated chloride secretion is preserved in respiratoryepithelia of CF patients compared to unaffected individuals, but issignificantly reduced or absent from CFTR-defective epithelia. Theseresults strongly suggest that an alternative non-CFTR regulated chloridechannel activity might account for attenuating CF disease in sometissues. Thus, a need exists for identification, isolation andfunctional analysis of alternative chloride channels.

Adhesion Molecules

It is apparent that endothelial cell adhesion molecules may havefunctions in addition to their adhesive functions. For example,integrins have transmembrane signalling capacities which may play a rolein the adherence process. However, the primary function of endothelialcell adhesion molecules is adherence to a substrate such as (a) topromote adherence of endothelial cells to basement membrane, (b) topromote vascular arrest and to facilitate extravasation of leukocytessuch as during an immune response, and (c) to promote homing oflymphocytes to a particular lymphoid tissue. Other molecules may play arole in controlling adherence of endothelial cells. For example,chloride ion channels are thought to be involved in a signalling cascadewhen lymphatic endothelial cells begin to adhere to a substrate (Martinet al., 1996, Microvasc. Res. 52:200-9).

There is considerable evidence that metastatic nonlymphoid tumor cellsmimic leukocytes in recognizing and adhering to one or more endothelialcell adhesion molecules to migrate in blood vessels, to arrest invascular areas of organs which may provide the microenvironmentconducive for metastatic growth, and to extravasate into surroundingtissues. An example of such an endothelial cell adhesion molecule whichpromotes adhesion of tumor cells and mediates metastasis islung-endothelial cell adhesion molecule (Lu-ECAM-1). Lu-ECAM-1 is a 90kilodalton (kDa) integral membrane protein constitutively expressedprimarily in endothelial cells of pleural and subpleural microvessels.Both in vitro studies and in vivo studies indicate thatLu-ECAM-1-expressing endothelial cells promote adhesion of certainlung-colonizing tumor cells in a manner that is consistent with theexpression level of the adhesion molecule and the metastatic propensityof tumor cells. For example, in an in vitro tumor cell/endothelial celladhesion assay, highly lung-metastatic B16-F10 melanoma cells bind tolung-matrix-modulated endothelial cells expressing Lu-ECAM-1 insignificantly larger numbers than their intermediate or lowlung-metastatic counterparts (B16-L8-F10 and B16F0, respectively; Zhu etal., 1991, Proc. Natl. Acad. Sci. USA 88:9568-720). Such binding appearsto be calcium (Ca²⁺) dependent. Further, anti-Lu-ECAM-1 monoclonalantibodies significantly inhibit adhesion of B16F10 melanoma cells toLu-ECAM-1 expressing endothelial cells in culture (Zhu et al., 1991,supra). Anti-Lu-ECAM-1 monoclonal antibodies are also efficient inpreventing metastatic colonization of the lungs by highlylung-metastatic B16F10 cells in a standard animal model for metastasis(Zhu et al., 1991, supra). Lu-ECAM-1, affinity purified from detergentextracts of bovine aortic endothelial cells, was used to immunize mice.The immunized mice showed an inhibition of metastatic colonization ofthe lungs by B16F10 melanoma cells, the efficiency of which wasdependent upon the anti-Lu-ECAM-1 serum titer (Zhu et al., 1992, J.Clin. Invest. 89:1718-1724). Lu-ECAM-1 appears to be the endothelialcell adhesion molecule for metastatic tumor cells that express theligand β4 integrin subunit (and possibly other ligands) including, butnot limited to, lung-metastatic breast tumor cells, and lung-metastaticmelanoma tumor cells.

Anti-adhesion therapy may be used to interfere with adhesion betweenorgan-specific endothelial cells and blood-borne cancer cells inpreventing the formation of metastatic colony formation in organs thatsupport metastatic cell growth. The amount of endothelial cell adhesionmolecule that can be made from detergent extracts, as well as the rateof production of the endothelial cell adhesion molecule, is generallyinsufficient for cost-effective commercial production. More efficientproduction of proteins, with a concomitant reduction in production cost,can often be achieved by producing a protein through recombinant means.In that regard, in some cases a host cell may be genetically engineeredsuch that an increased amount of the protein is produced and/or theprotein is produced in a manner which facilitates its isolation (ascompared to harvesting the protein from cell membranes).

SUMMARY OF THE INVENTION

It is an object of the invention to provide nucleotide sequences,isolated from mammalian endothelial cells, which encode molecules thatfunction as a calcium activated chloride channel-adhesion molecule(CACC-AM).

It is also an object of the present invention to provide nucleotidesequences which are variants (including portions) of the gene comprisingthe CACC-AM, and which encode a polypeptide having substantially thebiological activity as compared to the biological activity of theCACC-AM.

It is an object of the present invention to provide a means forrecombinantly producing CACC-AM molecule.

It is an object of the present invention to provide a means forrecombinantly producing proteins associated with CACC-AM molecule.

It is a further object of the present invention to provide expressionvectors containing a nucleotide sequence that encodes a CACC-AMmolecule; or containing a nucleotide sequence which is a variant of thegene for CACC-AM, and that encodes a polypeptide having substantialbiological activity of a CACC-AM; or containing a nucleotide sequencethat encodes a protein associated with a CACC-AM.

It is an additional object of the present invention to providerecombinant host cells which contain multiple copies of a nucleotidesequence that encodes a CACC-AM molecule, wherein the CACC-AM moleculeis recombinantly produced by culturing the recombinant host cells undersuitable conditions.

Other objects, features, and advantages of the present invention willbecome apparent from the following drawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a method for identifying clones usingpolymerase chain reaction. Also shown are restriction enzyme sites EcoRI(“R”); NdeI (“N”), PstI (“P”), and BglII (“B”).

FIG. 2A is a representation of immunoblots of bovine aortic endothelialcell proteins using either monoclonal antibody D3 (“D3”), polyclonalantibody CU11 (“11”), polyclonal antibody CU19 (“19”), polyclonalantibody R4 (“R4”), and polyclonal antibody R41 (“R41”).

FIG. 2B is a representation of Lu-ECAM-1 untreated (“−”) or Lu-ECAM-1treated with N-glycosidase F (“+”) followed by immunoblot analysis usingpolyclonal antibody R4; and Lu-ECAM-1-associated proteins untreated(“−”) or Lu-ECAM-1-associated proteins treated with N-glycosidase F(“+”) followed by immunoblot analysis using polyclonal antibody R41.

FIG. 3A is a representation of bovine aortic endothelial cells eitheruntreated (“−”) or treated with a crosslinker (“+”) followed byimmunoblot analysis using either polyclonal antibody R4 (“R4”), orpolyclonal antibody R41 (“R41”).

FIG. 3B is a representation of bovine aortic endothelial cells whichwere surface-biotinylated in the absence of (“−”) or presence of (“+”) acrosslinker followed by detection with streptavidin-horseradishperoxidase.

FIG. 4A is a representation of a ethidium bromide stained agarose gelcontaining the results of reverse transcriptase polymerase chainreaction analysis of bovine aortic endothelial cells (“BAEC”), lungtissue, tracheal epithelium, and spleen tissue using Lu-ECAM-1 specificprimer pairs L1, and L2.

FIG. 4B is a representation of a ethidium bromide stained agarose gelcontaining the results of reverse transcriptase polymerase chainreaction analysis of bovine aortic endothelial cells (“BAEC”), lungtissue, tracheal epithelium, and spleen tissue using bovine trachealchloride channel (“Ca-CC”) specific primer pairs T1, and T2.

FIG. 5 is a bar graph illustrating lung-metastatic tumor cell adhesionto wild type Lu-ECAM-1 in the presence or absence of anti-Lu-ECAM-1 mAb6D3; and lung-metastatic tumor cell adhesion to recombinant Lu-ECAM-1 inthe presence or absence of anti-Lu-ECAM-1 mAb 6D3.

FIG. 6 is a representation of the expression of mCLCA1 by in vitrotranslation (A) and in transfected HEK293 cells (B).

FIG. 7 is a representation of the biochemical analysis of hCLCA1 proteinfor in vitro translated (a), c-myc tagged hCLCA1 transfected HEK293 (b),and surface expression of c-myc tagged hCLCA1.

FIG. 8 is a representation of biochemical analysis of the hCLCA2 proteinfor in vitro translation (a) and immunoblot detection of myc taggedhCLCA2 constructs in HEK293 cells (b).

FIG. 9 is a representation of whole cell currents in mCLCA1-transfectedHEK293 cells.

FIG. 10 is an illustration of the summary the effects of inhibitors onmCLCA1 current expression.

FIG. 11 is a representation of whole cell currents in hCLCA1-transfectedHEK293 cells.

FIG. 12 is an illustration of the summary the effects of inhibitors onhCLCA1 current expression.

FIG. 13 is an illustration of the electrohpysiological analysis ofhCLCA2.

FIG. 14 is a representation of a comparison of the amino acid sequencesof the calcium activated chloride channels, hCLCA1 (SEQ ID NO:27);hCLCA2 (SEQ ID NO:31); bCLCA1 (SEQ ID NO:46); LU-ECAM-1 (SEQ ID NO:1);and mCLCA1 (SEQ ID NO:33).

DETAILED DESCRIPTION

Definitions

“Precursor” is a term used in conjunction with “lung-endothelial celladhesion molecule” hereinafter for the purposes of the specification andclaims to refer to a sequence of amino acids bound to and locatedupstream from the N-terminal portion of the mature form of alung-endothelial cell adhesion molecule, wherein the removal of thissequence results in the formation of the “mature form” of thelung-endothelial cell adhesion molecule. A precursor protein is a formof a lung-endothelial cell containing a prepro-region. The prepro-regionis made up of amino acids comprising a signal sequence, wherein thesignal sequence is cleaved to form the mature form of a lung-endothelialcell adhesion molecule.

“Calcium activated chloride channel-adhesion molecule” or “CACC-AM” is aterm used hereinafter for the purposes of the specification and claimsto mean a molecule isolated from mammalian endothelial cells that whenexpressed in cells induces the expression of calcium activated chlorideconductance channels.

“Calcium activated chloride channel(s)” is a term used for the purposesof the specification and claims to mean chloride channels whoseconductance is activated by calcium as judged by inhibition ofconductance by DIDS, DTT or niflumic acid.

“Recombinant calcium activated chloride channel-adhesion molecule” or“Recombinant CACC-AM” is a term used hereinafter for the purposes of thespecification and claims to refer to a CACC-AM molecule produced from aheterologous cell (e.g., other than from vascular endothelial cells),wherein the heterologous cell has been genetically engineered to containa nucleotide sequence that encodes a CACC-AM molecule.

“Recombinant calcium activated chloride channel-adhesionmolecule-associated protein” or “recombinant CACC-AM-associatedmolecule” is a term used hereinafter for the purposes of thespecification and claims to refer to a CACC-AM associated proteinproduced from a heterologous cell (e.g., other than from vascularendothelial cells) wherein the heterologous cell has been geneticallyengineered to contain a nucleotide sequence that encodes a CACC-AMassociated molecule. “Lung-endothelial cell adhesion molecule-associatedprotein” is a term used hereinafter for the purposes of thespecification and claims to refer to a protein which (a) is smaller inkilodaltons than the mature form of the lung-endothelial cell adhesionmolecule, as determined by, for example, sodium dodecyl polyacrylamidegel electrophoresis (SDS-PAGE) or amino acid analysis; (b) is encoded bymessages that also encode the lung-endothelial cell adhesion molecule;(c) is antigenically distinct from the lung-endothelial cell adhesionmolecule; and (d) is extracellularly associated in a complex (e.g,specific binding) with the lung-endothelial cell adhesion molecule.

By the term “operably linked” is meant, for the purposes of thespecification and claims to refer to the chemical fusion (restrictionwith subsequent ligation) or synthesis of heterologous DNA with anucleotide sequence that encodes a lung-endothelial cell adhesionmolecule or a lung-endothelial cell adhesion molecule-associated proteinsuch that the resultant recombinant DNA molecule is formed in a properorientation and reading frame for the nucleotide sequence to betranscribed into functional RNA. In the construction of the recombinantDNA molecule, it is generally preferred to position a promoter at adistance upstream from the initial codon of the nucleotide sequence thatis approximately the same as the distance in its natural setting (e.g.,in an endothelial cell). However, as known in the art, some variation inthe distance can be accommodated without loss of promoter function.Likewise, it is generally preferred to position an enhancer element at adistance upstream from the promoter, or incorporated into the promotersequences as a promoter element, or located between the promoter and theDNA molecule to be expressed. However, as known in the art, somevariation in the placement can be accommodated without loss of theenhancer element's function. “Expression control sequences” is meant,for the purposes of the specification and claims to refer to a promoteror promoter-enhancer combination.

By the term “expression vector” is meant, for the purposes of thespecification and claims to refer to a DNA molecule which is operablylinked to a nucleotide sequence that encodes one or more recombinantproteins comprising a lung-endothelial cell adhesion molecule and/or alung-endothelial cell adhesion molecule-associated protein such that theproduction of the recombinant protein is effected in a suitable host.The vector may include, but is not limited to, a plasmid, phage, or apotential genomic insert.

By the terms “degeneracy substitutions”, for the purposes of thespecification and claims to refer to the base pair changes(substitutions) in the nucleotide sequence such as a change in one ormore bases of a triplet codon (e.g., third base degeneracy) resulting inthe encoding of the same amino acid as before the change, or a changeresulting in the encoding of a conservative substitution in the aminoacid sequence encoded. With respect to such variations, and asappreciated by those skilled in the art, because of third basedegeneracy, almost every amino acid can be represented by more than onetriplet codon in a coding nucleotide sequence. Thus, in nature or bymutagenic means, the nucleotide sequence be modified slightly insequence (e.g., substitution of a nucleotide in a triplet codon), andyet still encode its respective gene product of the same amino acidsequence as encoded by the disclosed nucleotide sequences.

Further, the nucleotide sequence may have minor base pair changes whichmay result in variation (conservative substitution) in the amino acidsequence encoded. Such conservative substitutions are not expected tosubstantially alter the biological activity of the gene product. A“conservative substitution” for the purpose of the specification andclaims means modification of one or more amino acids are such that thetertiary configuration of the recombinant protein is substantiallyunchanged. Conservative substitutions is defined by aforementionedfunction, and includes substitutions of amino acids having substantiallythe same charge, size, hydrophilicity, and/or aromaticity as the aminoacid replaced. Such substitutions, known to those of ordinary skill inthe art, include glycine-alanine-valine; isoleucine-leucine;tryptophan-tyrosine; aspartic acid-glutamic acid; arginine-lysine;asparagine-glutamine; and serine-threonine. It is noted that anucleotide sequence according to the present invention encodes amammalian Lu-ECAM-1, as to be described more fully herein, and does notencompass the nucleotide sequence encoding the bovine trachealepithelial chloride channel described recently (Cunningham et al., 1995,J. Biol. Chem. 270:31016-26).

By the terms “% identity of amino acid sequence” are meant, for thepurposes of the specification and claims to refer to the percent ofamino acid positions that are identical between two amino acid sequencesas determined by sequence comparisons performed using algorithms knownto those skilled in the art.

By the terms “% identity of nucleotide sequence” are meant, for thepurposes of the specification and claims to refer to the percent ofnucleotide base pair positions that are identical between two nucleotidesequences as determined by sequence comparisons performed usingalgorithms known to those skilled in the art.

By the term “substantially” is used in conjunction with the biologicalactivity (e.g., adhesive function or chloride ion channel function) tomean, for the purposes of the specification and claims, to refer toretaining a degree of the biological activity ranging from approximately50% of the activity to greater than 100% of the activity, in relation tothe molecule with which it is compared.

By the term “unexpectedly improved” is used in conjunction with thebiological activity (e.g., adhesive function or chloride ion channelfunction) of a recombinant protein to mean, for the purposes of thespecification and claims, to refer to a degree of the biologicalactivity which is approximately greater or equal to 30% more biologicalactivity than that of the molecule to which it is compared, and whichimprovement in activity was unforeseen for this recombinant protein.

The present invention relates to nucleotide sequences and variantsthereof that encode a polypeptide which is a calcium activated chloridechannel and/or has adhesion properties. In accordance with thisinvention, nucleotide sequences encoding Lu-ECAM-1/mouse calciumactivated chloride channel (mCLCA), and human calcium activated chloridechannel molecules (hCLCA1, hCLCA2, and hCLCA3) are disclosed. Thenucleotide sequences have been derived from bovine aortic endothelialcells, from murine aortic endothelial cells, or from human endothelialcells. In one embodiment, a nucleotide sequence of the presentinvention, SEQ ID NO:1, contains sequences that encode either Lu-ECAM-1or Lu-ECAM-1-associated protein. From SEQ ID NO:1, the lung-endothelialcell adhesion molecule precursor is deduced to be approximately 905amino acids (SEQ ID NO:2). Cleavage of the signal peptide (amino acids−21 to −1 of SEQ ID NO:2) from the lung-endothelial cell adhesionmolecule precursor, and subsequent post-translational processing,results in a Lu-ECAM-1 of about 799 amino acids (amino acid 1 to aminoacid 799 of SEQ ID NO:2) and with a predicted molecular size ofapproximately 87 kDa. It was also discovered during the development ofthe invention that a SEQ ID NO:1 encodes a Lu-ECAM-1-associated protein(SEQ ID NO:3) which, depending on the glycosylation pattern, has anapparent molecular size (e.g., as determined by SDS-PAGE) ranging fromabout 22 kDa (little or no glycosylation present) to 38 kDa. Moreparticularly, SEQ ID NO:1 encodes Lu-ECAM-1-associated proteins ofapparent molecular size of about 38 kDa and of about 32 kDa. Further,these two LIEU-ECAM-1-associated proteins bind with Lu-ECAM-1 (aminoacid 1 to amino acid 799 of SEQ ID NO:2) in forming Lu-ECAM-1 complex.The mCLCA, human CLCA1, and human CLCA2 were then cloned and sequencedusing the Lu-ECAM-1 open reading frame as a probe.

In accordance with another embodiment of this invention, usingrecombinant techniques a nucleic acid molecule containing the nucleotidesequence encoding calcium activated chloride channel-adhesion moleculeis incorporated into an expression vector. The recombinant vector isintroduced into an appropriate host cell thereby directing theexpression of the sequence in that particular host cell. The expressionsystem, comprising the recombinant vector introduced into the host cell,can be used to produce recombinant CACC-AM, or associated proteins.According to the present invention, recombinant CACC-AM, a recombinantpolypeptide having CACC-AM activity, and/or recombinant CACC-AMassociated protein, can be purified by methods known in the artincluding ion-exchange chromatography, affinity chromatography, or otherchromatographic separation techniques.

Another embodiment of the present invention is a method for providingcalcium-activated chloride conductance channels to mammalian cells. Inmammalian cells in which the membrane chloride ion channels aredeficient in number or function (e.g., in airway epithelial cells ofcystic fibrosis patients), a method of providing to mammalian cells acalcium-activated chloride conductance channel, comprising CACC-AM or apolypeptide having CACC-AM activity, comprises administering directly tothe cells an expression vector. The expression vector contains a nucleicacid molecule operably linked to expression control sequences, whereinthe nucleic acid molecule encodes a CACC-AM, with the resultantexpression vector being introduced into the mammalian cell, and thecalcium-dependent chloride conductance produced in the mammalian cellscontaining the expression vector.

The bovine Lu-ECAM-1 complex appears to be expressed in lung, spleen,and aortic epithelial cells. The murine Lu-ECAM-1 complex appears to beexpressed in lung, trachea, spleen, mammary gland, intestine, uterus,epididymis, testis, pancreas, kidney, liver and skin. A first humanCLCA1 (hCLCA1) molecule (SEQ ID NO:28) appears to be expressed in smallintestine, and colon mucosa. A second human CLCA2 (hCLCA2) molecule (SEQID NO:32) appears to be expressed in trachea and mammary gland. A thirdhuman CLCA3 (hCLCA3) molecule (SEQ ID NO:30) appears to be expressed insmall intestine, trachea, mammary gland, stomach, bone marrow, spleen,lymph node, and peripheral blood leukocytes. That these variousmammalian proteins appear to be expressed in tissues which are affectedin cystic fibrosis may allow them to be used as chloride channels inaccordance with Example 8 herein.

For purposes of the description, the following embodiments illustratethe manner and process of making and using the invention and set forththe best mode contemplated by the inventor for carrying out theinvention, but are not to be construed as limiting.

EXAMPLE 1

This embodiment illustrates the molecular cloning of calcium activatedchloride channel-adhesion molecules.

Lu-ECAM-1

A nucleic acid molecule encoding Lu-ECAM-1 and Lu-ECAM-1-associatedproteins according to the present invention can be obtained by preparingcDNA from total RNA isolated from a host cell expressing Lu-ECAM-1. Toillustrate this example, total RNA was isolated from bovine aorticendothelial cells by the guanidinium chloride procedure, and a Lu-ECAM-1CDNA clone was constructed using nucleic acid amplification assummarized in FIG. 1. First, the N-terminal and internal amino acidsequences of a 38 kDa Lu-ECAM-1-associated protein (SEQ ID NO:3) wereused to design degenerate primers for primary and nested polymerasechain reactions using the reverse-transcribed total RNA as template.Upstream primers corresponded to nucleotide sequences encoding aminoacids 685 to 693, and amino acids 698 to 705, of SEQ ID NO:3. Downstreamantisense primers corresponded to nucleotide sequences encoding aminoacids 839 to 832, and amino acids 852 to 846, of SEQ ID NO:3. A productof approximately 450 bp was amplified (illustrated in FIG. 1 as “P1”).From these sequences, nondegenerate primers (SEQ ID NOs: 4 and 5) weredesigned, and the resultant amplification for 3′ sequences resulted in aproduct of approximately 750 bp (FIG. 1, “P2”). Nondegenerate primers(SEQ ID NOs: 6 and 7) were designed, and the resultant amplification for5′ sequences resulted in a product of approximately 1000 bp (FIG. 1,“P3”). To obtain the remaining 5′ sequences (FIG. 1, “P4”) including asignal sequence and the ATG initiation codon, used was an internalprimer (SEQ ID NO:8). To reconstitute the CDNA sequence from theamplified products (Pi-P4), the overlapping products were assembled intoone open reading frame by an over-lap extension strategy using a highfidelity polymerase combination. The result was clone 1 (FIG. 1)comprising 3.3 kb and encoding the amino acid sequence of SEQ ID NO:2.Hydrophilicity analysis revealed six significant generally nonpolarregions. In particular, a hydrophobic sequence from amino acid 595 toamino acid 618 appears to be a transmembrane domain. Nine potentialsites exist for asparagine-linked glycosylation.

Using the primers to probe a lambda cDNA library, three additionalclones (clones 2, 3, and 4; FIG. 1) were identified and sequenced.Additional primers (SEQ ID NOs: 9 and 10) were used to obtain the 5′ endsequences. Clone 2, a 3.3 kb variant of clone 1, was identical to clone1 from nucleotide 252 to nucleotide 2438 of SEQ ID NO:1, but then thesequence diverged. The amino acid sequence deduced from clone 2 (SEQ IDNO:11) was identical to that of clone 1 up to amino acid 772 (of SEQ IDNO:2) followed by a glutamate and serine. Clone 3 was 2.8 kb variant ofclone 1. The amino acid sequence deduced from clone 3 (SEQ ID NO:12) wasidentical to that of clone 1 up to amino acid 772 (of SEQ ID NO:2),followed by an additional 28 amino acids. Clone 4, of 1.3 kb, appears toencode a truncated 321 amino acid (SEQ ID NO:13) variant of Lu-ECAM-1that may be secreted, and is identical in sequence to amino acids 1 to303 of SEQ ID NO:2, followed by 18 divergent amino acids. Anoligonucleotide probe (SEQ ID NO:14) synthesized from the unique 3′region of clone 1 was used to hybridize MRNA isolated from bovine aorticendothelial cells. The probe detected high molecular weight bands (6-10kb) in Northern blot analysis as well as the 3.3 kb band. However, theprobe did not hybridize to the 2.8 and 1.3 kb bands. These resultsindicate that the 38 kDa and 32 kDa proteins appear to be encoded onlyby the messages that also encode the 90 kDa protein.

This embodiment also illustrates that CACC-AM is conserved in mammalianspecies, and thus may serve the same or similar functions in mammalianspecies other than the ones disclosed herein. Conservation of the geneencoding CACC-AM was determined by multispecies genomic DNA (from human,green monkey, rat, mouse, dog, bovine, rabbit, chicken, and buddingyeast) blot with probes derived from various regions of the bovine cDNAsequence for Lu-ECAM-1. These probes hybridized to all mammalian speciesgenomic DNA, although the hybridization to rat DNA was comparativelyweak. No hybridization signal was detected for chicken DNA or yeast DNA.These results indicate that the gene (or variant sequence thereof)encoding Lu-ECAM-1 is highly conserved in mammalian evolution.

Accordingly, using similar methods and primer sequences for isolatingand sequencing of a nucleotide sequence encoding a bovine Lu-ECAM-1,various nucleotide sequences encoding other CACC-AMs may be identified.

Mouse Calcium Activated Chloride Channel

As an illustration, a murine CACC/AM has been identified. A mouse lungcDNA library in lambda-gt11 was screened with the open reading frame ofLu-ECAM-1 cDNA (EcoR1-BglII 2.4 kb fragment of the Lu-ECAM-1 cDNA) usinglow stringency hybridization conditions (hybridization at 65 C. in5×SSC, 5×Denhardt's solution and 0.2% SDS solution overnight withagitation; washing with 2×SSC followed by several washes in 0.2×SSC,0.2% SDS at room temperature for a total of 30 minutes). Positive phageswere purified and analyzed by Southern blot hybridization techniques.Standard sequencing techniques (eg. automatic sequencing techniques)were used to determine the sequence of the clones. The largest of theisolated CDNA was 2.2 kb in length. It lacked the 5′ end as determinedby sequence comparison with the known bovine homolog. A full lengthmouse Lu-ECAM-1 was constructed by amplification of the 5′ cDNA endsfrom a pool of mouse lung poly(A)+RNA (CLONTECH). A gene-specific primer(SEQ ID NO:35) was used to reverse transcribe the cDNA from mouse lungmRNA. A nested primer (SEQ ID NO:36) and a primer recognizing the5′terminal tag were used to amplify the 5′ end of the cDNA by polymerasechain reaction. PCR products were cloned into an expression vector(pGEM-3; Promega). A full length mouse mCLCA1 was assembled by fusingthe rapid amplification product clone with the 2.2 cDNA insert in anexpression vector (pmlI site of pBluescript, Stratagene). Thus a 3.02 kblong sequence (SEQ ID NO:33) encoding a polypeptide of 902 amino acids(SEQ ID NO:34) was obtained.

Human CLCA1

In another illustration, a nucleic acid molecule encoding human calciumsensitive chloride channels was obtained from either the genomic libraryor a cDNA library. A human genomic library was screened with the ORF ofbovine Lu-ECAM-1 as probe using standard plaque hybridizationtechniques. Three positive clones of 4,6, and 7 kb were isolated andsequenced, spanning a contiguous genomic fragment of 14 kb withinterspersed segments of 58 to 65% nucleotide identity to parts of theLu-ECAM-1 ORF. Since the regions of homology did not encode a contiguousopen reading frame and did not cover the entire Lu-ECAM-1 ORF, theremaining parts of the gene were obtained by genomic walking usingnested PCR primers from each 5′ and 3′ end of the clones obtained byplaque hybridizations. Nested PCR conditions were 20 cycles for thefirst amplification step and 30 cycles for the second amplification withannealing temperatures of approximately 2° C. below the calculatedmelting point of the primers and extension times of 5 min per cycle. PCRproducts were cloned into a vector (pGem-T, Promega) and sequenced. Thefull length gene was isolated and sequenced spanning 31,902 bp. Thereading frame of the genomic sequence was determined according to itssequence homology with bCLCA1, Lu-ECAM-1 and mCLCA1.

Using an RT-PCR based strategy, the CLCA1 cDNA was cloned and sequencedfrom small intestinal mRNA. PCR primers (downstream primer SEQ ID NO:37,and upstream primer SEQ ID NO:38) flanking the ORF and containinglinkers with NotI restriction sites were generated and used to amplifythe 2745 bp ORF. RT-PCR was performed with 500 ng of human smallintestinal poly(A+) (CLONTECH). Reverse transcription was carried out at48° C. with Superscript RNase H-reverse transcriptase and PCR wasperformed with Pwo DNA polymerase (Boehringer). PCR conditions were asfollows: initial denaturation at 94° C. for 3 min followed by additionof DNA polymerase; 35 cycles of 94° C. for 50 s, 58° C. for 30 s, and72° C. for 2 min with a time increment of 3 s per cycle for eachextension step, followed by a final extension step of 72° C. for 8 min.Foe obtaining the untranslated region of CLCA1 mRNA, amplification ofthe 5′ and 3′ ends was carried out using primers SEQ ID NO:39 and SEQ IDNO:40 respectively. The resulting CDNA sequence (SEQ ID NO:27) comprises3007 bp and is identical to the genomic fragments with high sequencesimilarity to the previously cloned homolog. It contains a single ORF of2745 bp encoding a polypeptide of 914 amino acids (SEQ ID NO:28).

hCLCA2 cDNA

A human lung cDNA library (Clontech) was screened using Lu-ECAM-1 cDNAas probe as described above. Missing 5′ and 3′ ends of the isolated cDNAspecies were completed using RACE (Life Technologies). A single 3.6 kbcDNA species was identified and termed CLCA2. A sequence of 2970 bp isshown in SEQ ID NO:31. The open reading frame of The nucleotide sequenceencoding a polypeptide of 943 amino acids (SEQ ID NO:32) shared highdegrees of identity with those of Lu-ECAM-1 (86%), bCLCAl (85%), mCLCAl(76%), and hCLCAI (63%)—FIG. 14.

hCLCA3 cDNA

A human spleen CDNA library packed in phage λgt11 (Clontech) wasscreened using standard plaque hybridization protocols. The open readingframe (ORF) of the Lu-ECAM-1 cDNA was used as probe as described above.Phage colony blots were hybridized and washed at low stringencyconditions (hybridization: 55° C. overnight in 4×SSC standardhybridization buffer without formamide; two stringency washes with2×SSC, 0.1% SDS at room temperature, and two washes with lxSSC, 0.1% SDSat 40° C.). After exhaustive screening of the library (>7×10⁶ plaques),a single positive phage clone was plaque-purified, amplified, andsubjected to DNA purification (Wizard Lambda Preps, Promega). The insertwas cut out using the EcoRI sites and cloned into pbluescript II SK(Stratagene). Automated sequencing with initial plasmid-derived primersfollowed by internal gene-specific primers was performed by the CornellUniversity DNA Sequencing Facility using dRhodamine Terminator CycleSequencing on an ABI Prism 377 DNA Sequencer (PE Applied Biosystems).Missing 5′ and 3′ ends of the cDNA were isolated using the rapidamplification of cDNA ends (RACE) technique (Life Technologies) andhuman spleen poly-A+RNA (Clontech) as template. The primers foramplification of 5′ end were SEQ ID NO:43 and SEQ ID NO:44, and theprimers for 3′ end was SEQ ID NO:45. The resulting cDNA sequence of 3599base paris (deposited in GenBank under accession no. AF043976) wasobtained. A sequence of 3418 bp is shown in SEQ ID NO:29, which encodesfor a polypeptide of 1000 amino acids (SEQ ID NO:30).

EXAMPLE 2

This example illustrates the proteins encoded by the cDNAs isolated inExample 1 and the relationship between CACC-AM and associated proteins.As an illustration, the relationship is between Lu-ECAM-1 and Lu-ECAM-1associated protein is demonstrated. Antigenic characterization wasperformed by generating anti-Lu-ECAM-1 antibodies, and testing theantibodies in Western blot analyses of bovine aortic endothelial cellextracts. Rats were immunized with either the 90 kDa band excised from apolyacrylamide gel and mixed with adjuvant, resulting in polyclonalantibody R4; or a 38 kDa band excised from a polyacrylamide gel andmixed with adjuvant, resulting in polyclonal antibody R41. Two peptides(SEQ ID NOs: 15 and 16) were synthesized, conjugated to KLH, and used toimmunize rabbits in forming polyclonal antibodies CU11 and CU8,respectively. Monoclonal antibody 6D3 has binding specificity toLu-ECAM-1 as described previously (Zhu et al., 1992, supra).

As shown in FIG. 2A, mAb 6D3 detected a 90 kDa component (Lu-ECAM-1) andtwo larger bands of approximately 120 kDa and 130 kDa (Lu-ECAM-1precursors); but not the 38 kDa or the 32 kDa components(Lu-ECAM-1-associated proteins). Likewise, polyclonal antibody (againstamino acid residues of SEQ ID NO:15) recognized only the 90 kDa, 120kDa, and 130 kDa components (FIG. 2A). In contrast, polyclonal antibodyCU19 (against amino acid residues 618 to 767 of SEQ ID NO:2) stronglydetected the 38 kDa and 32 kDa components, and the 120 kDa and 130 kDacomponents, but only weakly detected the 90 kDa component. These resultsare evidence that the initial translation products of the open readingframe in SEQ ID NO:1 are the 120 kDa and 130 kDa components, which arethen processed to yield the 90 kDa, 38 kDa, and 32 kDa components.

These results were confirmed with polyclonal antibodies R4 and R41. R4,a polyclonal anti-90 kDa protein antibody, detected the 90 kDa band, aswell as the 120 kDa and 130 kDa components; but not the 38 kDa, and 32kDa components (FIG. 2A). R41, a polyclonal anti-38 kDa proteinantibody, detected the 38 kDa and 32 kDa bands, as well as the 120 kDaand 130 kDa components; but not the 90 kDa component (FIG. 2A). Theseresults indicate that (a) the 38 kDa and 32 kDa bands are antigenicallyrelated; (b) the 120 kDa and 130 kDa bands are antigenically related;and (c) the 120 kDa and 130 kDa bands have sequence in common with boththe 90 kDa protein, and the 38 kDa and 32 kDa proteins. Treatment ofLu-ECAM-1 complex with Nglycosidase F reduced the 38 kDa and 32 kDacomponents to a common band of about 22 kDa, indicating the these twoproteins represent alternate glycoforms (FIG. 2B). N-glycosidase Ftreatment reduced the 90 kDa protein to 77 kDa (FIG. 2B). The 77 kDa and22 kDa products would add up to the exact size of the initialtranslation product of clone 1 before processing.

As shown in FIG. 2A, the 38 kDa and the 32 kDa components of theLu-ECAM-1 complex are not recognized by mAb 6D3 in SDS-PAGE and Westernblot analysis, suggesting that these components are likely noncovalentlycomplexed with the 90 kDa protein. The Lu-ECAM-1 complex is resistant todissociation by high salt, detergent, and EDTA, but readily dissociateswhen boiled in SDS in the presence or absence of reducing agents (e.g.,dithiothrietol). To visualize the Lu-ECAM-1 complex, and to determinewhether the proteins of the complex are associated intracellulary orextracellularly, the surface of bovine aortic endothelial cells wascross-linked. Confluent bovine aortic endothelial cells were surfacebiotinylated in the presence or absence of disuccinimidyl tartarate(DST), a reagent that restricts cross-linking to extracellular moietiesof proteins in close contact. DST dissolved in dimethyl sulfoxide wasadded to the cells in a final concentration of 1 mM. Cross-linking wascarried out at 4° C. with gentle shaking. The reactions were stopped byadding glycine to a final concentration of 50 mM. After quenching for 5minutes, the cells were lysed for 1 hour in lysis buffer. Lysates wereclarified by centrifugation, precipitated with mouse-IgG agarose beads,then immunoprecipitated with mAb 6D3. Immunoprecipitated proteins wereanalyzed by SDS-PAGE, transferred to nitrocellulose, and detected usingavidin-horseradish peroxidase and chemiluminescence. As shown in FIG.3A, immunoblots using either R4 (polyclonal anti-90 kDa proteinantibody) or R41 (polyclonal anti-38 kDa protein antibody) detected anovel band migrating at approximately 140 kDa (arrow, FIG. 3A), with aconcomitant reduction in intensities of the 90 kDa, 38 kDa, and 32 kDacomponents. As illustrated in FIG. 3B, all Lu-ECAM-1 complex componentswere biotinylated on bovine aortic endothelial cell surface. Theseresults suggest that the Lu-ECAM complex is made up of either the 90 kDaand 38 kDa proteins complexed in an extracellular association, and/orthe 90 kDa and 32 kDa proteins complexed in an extracellularassociation.

In another illustration of this embodiment, the mCLCA1 protein wascharacterized. An in vitro transcription and translation system (TNT™,Promega) was used for the in vitro expression of the full length cDNA(SEQ ID NO:33). Canine microsomes were used to glycosylate the productof in vitro translation. In addition, HEK293 cells were transfected withthe cDNA of mCLCA1 using standard methods known to those skilled in theart (CaPO₄ or Lipfectamine, Life Technologies). Products were analyzedon SDS-PAGE gels. In addition, mCLCA1 cDNA was also used fortransfection of cells. Proteins prepared by standard in vitrotranslation techniques or from lysates of transfected HEK293 cells wereanalyzed on Western blotting by using rabbit polyclonal antibodiesagainst N-terminal (CU8) and the C-terminal region (CU21) of Lu-ECAMpeptide. As shown in FIG. 6, protein bands of 130, 125, 90 kDa andtriplet bands of 32-38 kDa were detected in transfected cells. CU8reacted exclusively with the large sized bands of 90, 125 and 130 kDawhereas CU21 reacted with only the triplet of the smaller bands. Thisrecognition pattern is similar to that observed for Lu-ECAM-1 andsuggests that the ORF of mCLCA1 cDNA encodes a precursor protein,represented by alternate glycoforms of 125 and 130 kDa, that isposttranslationally processed into 90 kDa and 38/32 kDa components.

In another illustration of this embodiment, the hCLCA1 protein wascharacterized. The ORF of the hCLCA1 cDNA encodes a 914 amino acidprotein with a calculated molecular weight of 100.9 kDA. In vitrotranslation of human CLCA1 cDNA yielded a single protein ofapproximately 100 kDa, consistent with its calculated size (FIG. 7). Inthe presence of canine microsomes the Mr of the polypeptide shifted to125,000 indicating multiple glycosylations. Similar to Lu-ECAM-1 andmCLCA1, 37-40 kDa proteins were not detected in immunoblots of wholecell lysates but were coimmunoprecipitated with the 90 and 125 kDaprotein. To ascertain whether the 125 kDa hCLCA1 protein is processedinto 90 kDa and 30-40 kDa cleavage products in a manner similar toLu-ECAM-1, c-myc tags were inserted in five different hydrophilic siteswith high surface probability (m1-m5) and were overexpressed in HEK293cells (Cravchik et al., 1993, Gene 137:139-143). Immunoblots of wholecell lysates probed with anti-myc antibodies revealed proteins of 125and 90 kDa (FIG. 7b). However, immunoprecipitation of cell lysatesfollowing surface biotinylation indicated the presence of 37-41 kDaproteins similar to Lu-ECAM-1 and mCLCA1 (FIG. 7c).

In another illustration of this embodiment, the human CLCA2 protein wasanalyzed. The predicted size of the full length protein (104 kDa) isconsistent with the result of an in vitro translation assay yieldingprimary translation product of approximately 105 kDa (FIG. 8a). Toascertain wheter the CLCA2 protein is cleaved into two subunits inmammalian cells as reported for other CLCAs, two constructs weregenerated with a c-myc tag within the amino or carboxy terminusrespectively as described by Cravchik et al., 1993, Gene 137:139-143)and transfected into HEK293 cells. Immunoblots of cell lysates probedwith anti-myc antibody identified an 86 kDa protein when the tag wasinserted near the amino terminus (m1) and a 34 kDa protein when the tagwas inserted near the amino terminus (m2)—FIG. 8b.

EXAMPLE 3

Tissue Distribution

This example illustrates the tissue distribution of CACC-AM. As anillustration, the distribution of Lu-ECAM-1/Lu-ECAM-1 complex in therespiratory tree, as demonstrated by immunohistochemistry. Tissuesections were probed with anti-Lu-ECAM-1 antibodies. Formalin-fixedsections of bovine trachea were first denatured by boiling for tenminutes in 4M urea in a microwave oven, then probed with polyclonalantibody R4 (raised against denatured Lu-ECAM-1). The sections were thenincubated with donkey anti-rat IgG and avidin-peroxidase conjugate. Theperoxidase conjugate was detected using diamino-benzidine as substrate,and then the slides were counterstained with hematoxylin. Lung sectionswere prepared and probed with mAb 6D3 as previously described (Zhu etal., 1993, Int. J. Cancer 53:628-633) except that a biotinylatedsecondary antibody was used, followed by the avidin-peroxidaseconjugate, diamino-benzidine as substrate, and counterstaining withhematoxylin. The immunohistochemical analyses revealed thatLu-ECAM-1/Lu-ECAM-1 complex was expressed predominantly in endothelia ofsmall to medium-size venules of the lung, and in the respiratoryepithelia of bronchi and trachea. To confirm the distribution ofexpression of Lu-ECAM-1/Lu-ECAM-1 complex, and to distinguish it fromthat of the bovine epithelial chloride channel (“Ca-CC”) describedrecently (Cunningham et al., 1995, supra), nucleic acid amplificationwas performed using specific primers as described herein in Example 4.

Tissue distribution for other CACC-AMs of the present invention weredetermined by Northern blot analysis and RT-PCR. Human multiple tissueNorthern blots (Clontech) contained 2 μg poly-A+RNA per lane of thefollowing tissues: heart, brain, placenta, lung, liver, skeletal muscle,kidney, pancreas, spleen, thymus, prostate, testis, ovary, smallintestine, colon mucosa, peripheral blood leukocytes, stomach, thyroid,spinal cord, lymph node, trachea, adrenal gland, and bone marrow. Blotswere hybridized labeled fragments for respective cDNAs. To exclude crosshybridization of related family members, highly stringent washingconditions were employed following the hybridization (two washes with2×SSC, 0.1% SDS at 65° C. for 30 min, followed by two washes with0.2×SSC, 0.1% SDS at 65° C. for 30 min). RT-PCR was performed using theabove-mentioned conditions and primers to detect the cDNA fragments inpoly-A+RNA samples from human tissues. PCR products were analyzed on anethidium bromide stained agarose gel. To exclude amplification of aclosely related family member, the PCR products were cut out of the gel,cloned into the pGem-T vector, and partially sequenced. In all RT-PCRassays, negative controls were included with water instead of RNA astemplate in the reverse transcription. To control for RNA quality aswell as reverse transcription and PCR conditions, a fragment of EF-1amRNA was amplified as described.

A mouse multiple tissue Northern blot when probed with HindIII fragmentof mCLCA1 ORF revealed the presence of a 3.1 kb transcript in brain andspleen and transcripts of 5 kb and 3.1 kb in heart, lung, liver, andkidney.

For human CLCA1, a single mRNA species of 3.3 kb was detected inNorthern blot hybridizations in small intestine and colon mucosa.Similar results were obtained with RT-PCR.

hCLCA2 mRNA was detected in trachea and mammary gland, using the 2832ORF of hCLCA1. While CLCA2 was not detected in the lung by Northern blothybridization, the more sensitive RT-PCR revealed its expression in lungin addition to trachea and mammary gland suggesting a significantlylower expression level in the lung.

No signals were detected in any of the tissues tested on Northern blotsusing the 2817 cDNA of hCLCA3. However, by RT-PCR a fragment of thehCLCA3 cDNA could be amplified form all tissues tested, i.e. spleen,lung, trachea, thymus and mammary gland.

EXAMPLE 4

This example demonstrates that Lu-ECAM-1 and the bovine epithelialchloride channel (“Ca-CC”) described recently by (Cunningham et al.,1995, J. Biol. Chem. 270:31016-31026) are distinct molecules.

1. Genetic Similarity

Sequence alignment of the open reading frame of SEQ ID NO:1 with theCA-CC cDNA shows that the nucleotide sequences share 92% identity at theDNA level. Comparing the deduced amino acid sequence of Lu-ECAM-1 (SEQID NO:2) with that of CA-CC shows 88% identity at the amino acid level.However, the differences appear randomly distributed, and thus,Lu-ECAM-1 and CA-CC appear to represent products of different genes.

2. Subunit Differences

As shown in FIGS. 2A, 2B, 3A, and 3B, it is clear that the precursorLu-ECAM-1 is a protein with an apparent molecular size of either 120 kDaor 130 kDa. The precursor Lu-ECAM-1 gets processed to a 90 kDa Lu-ECAM-1protein, and to either a 38 kDa or 32 kDa Lu-ECAM-1-associated protein.In contrast, CA-CC is a 140 kDa multimeric complex that can be reducedto a band comprised of 38 kDa subunits in the presence of a reducingagent (Cunningham et al., 1995, supra). This difference in subunitstructure is further evidence that Lu-ECAM-1/Lu-ECAM-1 complex is aglycoprotein distinct from CA-CC.

3. Molecular Expression Differences

It is possible that immunohistochemical staining with polyclonalantibody to Lu-ECAM-1 could detect CA-CC if CA-CC shared across-reactive epitope with Lu-ECAM-1. To distinguish Lu-ECAM-1expression from CA-CC expression in tissues, reverse transcriptasepolymerase chain reaction was performed. Messenger RNA (500 ng) frombovine lung tissue, from bovine spleen tissue, from bovine trachealepithelium, and from cultured bovine aortic endothelial cells wasreverse-transcribed with random oligonucleotide primers and reversetranscriptase in a 20 μl reaction volume. Primers specific for Lu-ECAM-1sequences (primer pairs “L1”: SEQ ID NOS: 17 and 18, “L2”: SEQ ID NOs:19 and 20), and primers specific for CA-CC sequences (primer pairs“T1”:SEQ ID NOs: 21 and 22, and “T2” SEQ ID NOs: 23 and 24) wereconfirmed for selectivity by control experiments with a Lu-ECAM-1 cDNAclone. Amplification was performed using 1 μl of the respective cDNAsubstrate for 35 cycles of amplification in a reaction volume of 50 μlusing 0.5 units of thermostable DNA polymerase, 200 μM of each dNTP, 1,5mM MgCl₂, and 1 μM of the respective primer pair. The cycling protocolwas 94° C. for 20 seconds, 55° C. for 10 seconds, and 72° C. for 10seconds, with a time increment of 2 seconds per cycle for annealing andextension times. A final extension step was performed at 72° C. for 10minutes. Aliquots (5 μl) of each amplification reaction was fractionatedon a 1.5% agarose gel, and stained with ethidium bromide.

The calculated size for product amplified using primer pair L1 is 232bp; the calculated size for product amplified using primer pair L2 is218 bp; the calculated size for product amplified using primer pair T1is 231 bp; and the calculated size for product amplified using primerpair T2 is 218 bp. As shown in FIG. 4A, Lu-ECAM-1 is expressed in bovineaortic endothelial cells, lung tissue, and spleen, tissue, but not intracheal epithelium. In contrast, as shown in FIG. 4B, CA-CC isexpressed in lung tissue and tracheal epithelium, but not in bovineaortic endothelial cells nor spleen tissue. These results furthersupport that Lu-ECAM-1 and CA-CC are different molecular entities, withLu-ECAM-1 being expressed in venular endothelial cells, and CA-CC beingexpressed in tracheal and bronchial epithelial cells.

EXAMPLE 5

This embodiment illustrates that a nucleic acid molecule comprising anucleotide sequence encoding CACC-AM, or a variant sequence thereof, orencoding one or more CACC-AM associated proteins, can be inserted intovarious vectors including phage vectors and plasmids. Successfulexpression of the protein(s), requires that either the insert comprisingthe nucleotide sequence, or the vector itself, contain the necessaryelements for transcription and translation (expression control elements)which is compatible with, and recognized by the particular host systemused for expression. A variety of host systems may be utilized toexpress the recombinant protein(s), which include, but are not limitedto bacteria transformed with a bacteriophage vector, plasmid vector, orcosmid DNA; yeast containing yeast vectors; fungi containing fungalvectors; insect cell lines infected with virus (e.g. baculovirus); andmammalian cell lines transfected with plasmid or viral expressionvectors, or infected with recombinant virus (e.g. vaccinia virus,adenovirus, adeno-associated virus, retrovirus, etc.).

Using methods known in the art of molecular biology, including methodsdescribed above, various promoters and enhancers can be incorporatedinto the vector or the nucleic acid molecule encoding the recombinantprotease, to increase the expression of the recombinant protein(s),provided that this increased expression is compatible with (for example,non-toxic to) the particular host cell system used. The selection of thepromoter will depend on the expression system used. Promoters vary instrength, i.e. ability to facilitate transcription. Generally, for thepurpose of expressing a cloned gene, it is desirable to use a strongpromoter in order to obtain a high level of transcription of the gene orthe variant sequence and expression into the recombinant protein. Forexample, bacterial, phage, or plasmid promoters known in the art fromwhich a high level of transcription has been observed in a host cellsystem comprising E. coli include the lac promoter, trp promoter, tacpromoter, reca promoter, ribosomal RNA promoter, the PR and PLpromoters, lacUV5, ompf, bla, lpp, and the like, may be used to providetranscription of the inserted DNA sequence encoding the recombinantprotein.

As known to those skilled in the art, such vectors for expression inmammalian cells can be selected from plasmids, viruses, andretroviruses. For a recent review of vectors useful in gene therapy, seeWeichselbaum and Kufe (1997, Lancet, 349:S10-S12). The features of avector which make it useful in the methods of the present inventioninclude that it have a selection marker for identifying vector which hasinserted therein the nucleotide sequence to be expressed; restrictionsites to facilitate cloning; and the ability to enter and/or replicatein mammalian cells. Examples of a preferred vector for the in vivointroduction of a recombinant vector into mammalian cells include, butare not limited to viral vectors. Virus-based vectors are one preferredvehicle as they infect cells in vivo, wherein during the infectionprocess the viral genetic material is transferred into the cells. Aretroviral vector, such as a plasmid containing AAV (Adeno-associatedvirus) sequences, has been described previously (see for exampleChatterjee et al., 1992, Science, 258:1485-1488; U.S. Pat. No.5,252,479, herein incorporated by reference). Examples of other vectorsfor the in vitro or in vivo introduction into mammalian cells includeretroviral vectors (Miller et al., 1989, BioTechniques 7:980-990; Kormanet al., 1987, Proc. Natl. Acad. Sci. USA 84:2150-54), papovavirusepisomes (U.S. Pat. No. 5,624,820, herein incorporated by reference),and adenovirus vectors (U.S. Pat. No. 5,585,362, herein incorporated byreference). Promoters are known to those skilled in the art, and mayinclude viral or viral-like basal promoters like the SV40 late promoter,the RSV promoter, the CMV immediate early promoter, and a VL30 promoter;and cellular promoters (See, e.g., Larsen et al., 1995, Nucleic AcidsRes. 23:1223-1230; Donis et al., 1993, BioTechniques 15:786-787; Dondaet al., 1993, Mol. Cell. Endocrinol. 90:R23-26; and Huper et al., 1992,In Vitro Cell Dev. Biol. 28A:730-734).

In one illustration of this embodiment, a nucleotide sequence comprisingclone 1 (SEQ ID NO:1) was placed under the control of atetracycline-regulated promoter in a commercially available plasmid(pTet-Splice; GIBCO). The construction was accomplished in two steps. Anamplified product was generated that corresponded to the 3′ end of clone1 cDNA (nucleotide 2391 to nucleotide 2780 of SEQ ID NO:1) using a 5′primer containing an EcoRI restriction site (SEQ ID NO:25) and a 3′primer containing a SpeI restriction site (SEQ ID NO:26). The cyclingprotocol included 93° C. for 35 seconds, 55° C. for 60 seconds, 72° C.for 3 minutes for 40 cycles followed by a 10 minute incubation at 72° C.using a thermostable DNA polymerase. The product was cleaved with EcoRIand SpeI, then cloned into corresponding restriction sites in theplasmid. The resultant plasmid was selected and then sequenced toconfirm absence of mutations. This recombinant plasmid was then cleavedwith EcoRI and BglII. To reconstitute the open reading frame encodingLu-ECAM-1, the 2.3 kb EcoRI/BglII fragment was excised from clone 3 andinserted into the plasmid. The resulting plasmid, pTet-Splice-Lu-ECAM-1,was then co-transfected into HEK293 cells with another plasmid(pTet-tTAK) that encodes a transcriptional activator specific for thepTet-Splice vector. Transfection was done using a transfection reagent(lipofectamine) according to the manufacturers instructions. Cells wereharvested 24 hours after the start of transfection. Immunoblot analysisof the cells using polyclonal R41 resulted in the detection ofrecombinant Lu-ECAM-1 precursor of 120 kDa, and recombinantLu-ECAM-1-associated protein of 38 kDa. When the cells were probed inimmunoblot with anti-peptide antibody CU8, detected was recombinantLu-ECAM-1 precursor of 120 kDa, and recombinant Lu-ECAM-1 of 90 kDa.

In another embodiment of the invention, mCLCA1 cDNA was cut from thepBluescript vector (Stratagene) with SacI and PvuI, blunt ended withKlenow Polymerase and inserted into the tetracycline sensitive mammalianexpression vector (pTet-splice, Life Technologies, Inc.) at the EcoRVsite. HEK293 cells were cotransfected with mCLCA1 cDNA cloned into thepTet-splice alon with a vector expressing a tetracycline activator(pTet-tTak) using standard transfection techniques well known to thoseskilled in the art and as described above (Lipofectamine, LifeTechnologies, Inc.). Cells were cotransfected with a reporter vector asdescribed above. In another illustration of this embodiment, humanCLCA1, HEK293 cells were transfected with either pcDNA 3.1 containingthe CLCA1 insert and a reporter vector (enhanced green fluorescentprotein, EGFP, CLONTECH) or the reporter vector alone. Trnasfection canbe carried out by standard techniques known to those skilled in the artincluding CaPO4 precipitation or Lipofectamine (Life Technologies).

For human CLCA2, HEK293 cells were transfected using Lipofectamine usingmanufacturer's instructions. For example, 5 ul lipid and 0.5 ul of CLCA2were cloned into pcDNA 3.1 per 35 mm well in a 2-3 hour incubation. Forexpression studies, the 2,832 bp CLCA2 ORF was PCR amplified from humantrachea poly-A⁺ RNA (Clontech) following reverse transcription withSuperscript RNase H⁻ reverse transcriptase (Life Technologies) andrandom hexamer priming. PCR was performed with Pwo DNA Polymerase(Boehringer; initial denaturation at 94° C. for 3 min, 35 cycles of 940for 50 s, 58° C. for 30 s, and 72° C. for 2 min with a time increment of3 s per cycle for each extension step (72° C.), followed by a finalextension step of 72° C. for 8 min). Primer sequences were (upstreamprimer: SEQ ID NO:41, downstream primer: SEQ ID NO:42 with NotI-linkersunderlined). PCR products were gel purified, incubated with NotI, andcloned into the expression vector pcDNA3.1 (Invitrogen). Four differentPCR products were sequenced to control for potential PCR-inducedsequence errors. Cells were simultaneously cotransfected with a reportervector as described above. Chloride channel conductance activity wasrecorded after allowing the cells to recover for 24 hours.

The 2817 bp fragment of the hCLCA3 cDNA cloned into pcDNA3.1 wassimultaneously transcribed and translated as described for the otherCACC-AMs. Samples were analyzed by 10% SDS-PAGE (5 μl of a 25 μIreaction), followed by drying of the gel and exposure to film for 8 h.Protease protection assays were performed as described [10] to ascertainwhether hCLCA3 translation products were translocated into themicrosomes and thus entered the secretory pathway. In the presence ofmicrosomal membranes in vitro translated and ³⁵S-labeled wild typehCLCA3 was digested with Proteinase K (Sigma; 100 μg/ml) for 60 min onice with or without detergent present (0.5% Nonidet-P 40). The reactionwas stopped by adding phenylmethylsulfonyl fluoride and the productswere analyzed by 10% SDS-PAGE and exposure to film. To allow forimmunological detection of the translation products, three immunotaggedcDNA clones were constructed (m1 to m3) by inserting a partial sequenceof the human c-myc protein (EQKLISEEDL (SEQ ID NO:47)) [11] into theamino termini of the first (m1), the second (m2), or both (m3) ORFS.Generation of these constructs using overlap extension PCR and Pwo DNApolymerase (Boehringer) was as described [4]. Correct sequences of theconstructs were verified by sequencing. Immunotagged DNA constructs wereeither in vitro translated as described above or transfected into 70%confluent human embryonic kidney (HEK) 293 or chinese hamster ovary(CHO) cells via the Lipofectamine Plus method (Life Technologies). Celllysates were harvested after 48 h, resolved via 10% SDS-PAGE, andelectroblotted onto nitrocellulose. Blots were probed withmouse-anti-human c-myc antibody 9E10 (1 μg/ml; Calbiochem) as primaryantibody, horseradish peroxidase-conjugated goat anti-mouse antibody(0.2 μg/ml) as secondary antibody, and developed using enhancedchemiluminescence (Amersham). Secretion of the recombinant hCLCA3protein into the culture supernatant was assayed by concentrating theconditioned medium (24 to 48 h after transfection) of HEK 293 or CHOcells transfected with construct m3 using ultrafiltration devices with amolecular cutoff at 10 kDa (Ultrafree-15, Biomax-10 filter; Millipore;centrifugation at 2,000 g for 30 min at 4° C.).

EXAMPLE 6

This embodiment demonstrates that the CACC-AMs of the present inventioncan function as adhesion molecules. As an illustration, a recombinantLu-ECAM-1, encoded by a nucleic acid molecule according to the presentinvention, has unexpectedly improved biological activity. Recombinant(r) Lu-ECAM-1 and wild type (wt) Lu-ECAM-1 were compared in theiradhesion ability to lung-metastatic B16-F10 melanoma cells. Usinganti-Lu-ECAM-1 mAb 6D3, wtLu-ECAM-1 was purified from extracts of bovineaortic endothelial cells, and rLu-ECAM-1 was purified from extracts oftransfected HEK293 cells. The tumor cell adhesion assay was performed asdescribed previously (Zhu et al., 1992, supra). Briefly, 100 μg/ml inphosphate buffered saline of either wtLu-ECAM-1 or rLu-ECAM-1 was usedto coat wells of 96 plates overnight at 4° C. Wells were then washedwith tissue culture medium, and each well is seeded with a suspension oftissue culture medium and 2×10⁴ tumor cells which had beenradio-labelled. After being spun onto the coated wells at 15 g for 1minute, and incubated for 10 minutes at 37° C., nonadherent tumor cellswere spun off at 150 g for 5 minutes. Adherent tumor cells were thendissolved in 1% SDS and counted in a liquid scintillation counter. Tumorcell attachment is recorded as the percent cells bound of the totalcells seeded. Inhibition of tumor cell adhesion is determined by firstincubating the Lu-ECAM-1 coated wells with mAb 6D3 (1 μg/ml) for 1 hourat room temperature before the tumor cells are added.

As shown in FIG. 5, recombinant Lu-ECAM-1 has unexpectedly improvedbiological activity (e.g., adhesive function to lung-metastatic tumorcells) as compared to wild type Lu-ECAM-1. More particularly, rLu-ECAM-1supported adhesion of 87% of lung-metastatic tumor cells, whereaswtLu-ECAM-1 supported adhesion of only 43% of lung-metastatic tumorcells. Lung-metastatic tumor cell adhesion to wtLu-ECAM-1 was almostcompletely blocked by anti-Lu-ECAM-1 mAb 6D3, whereas lung-metastatictumor cell adhesion to rLu-ECAM-1 was only partially inhibited (66%) bythe concentration of anti-Lu-ECAM-1 mAb 6D3 used.

EXAMPLE 7

A comparison of the amino acid sequence of the CACC-AMs of the presentinvention is shown in FIG. 9. Sequence alignment and homology searcheswere carried out by using standard commercial software. For example,BLAST program was used for homology searches in existing data bases, andMegalign of the DNAStar package (Lasergene) was used for multiplesequence alignment. The sequence alignment of the four CACC-AMs of thepresent invention and the bovine CLCA (Cunningham et al. supra)indicates conservation throughout the entire length of the sequence,without the compartmentalization of more conserved domains. Nosignificant homologies to any other chloride channel proteins weredetected.

Table 1 illustrates a comparison of the size of the various mammalianLu-ECAM-1 proteins and Lu-ECAM-1 associated proteins as encoded by therespective open reading frames.

TABLE 1 Total # of Species SEQ ID NO: Amino Acids Predicted Size bovine2 and 3 905 a.a. 90 kD, 32-28 kD human 28 914 a.a. 90 kD, 40 kD hCLCA1human 30 1000 a.a. 130 kD hCLCA3 processing not known) human 32 943 a.a.130 kD hCLCA2 processing not known) murine 34 902 a.a. 130 kD, 125 kD,mCLCA 90 kD, 32-38 kD

Table 2 is a comparison among the mammalian Lu-ECAM-1 family showingboth an approximated amino acid similarity and an approximated aminoacid identity (expressed as “similarity/identity”).

TABLE 2 bovine murine human human human (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NOs: 2&3) NO: 34) NO: 28) NO: 30) NO: 32) bovine 100/100 81.3/67.4/ 85.7/ 63.7/ (SEQ ID 70.8 52.4 77.4 49.8 NOs: 2&3) murine — 100/10067.5/ 80.9/ 62.8/ mCLCA 52.7 69.5 48.4 (SEQ ID NO: 34) human — — 100/10065.3/ 62.3/ hCLCA1 51.4 44.7 (SEQ ID NO: 28) human — — — 100/100 62.1/hCLCA3 48.2 (SEQ ID NO: 30) human — — — — 100/100 hCLCA2 (SEQ ID NO: 32)

Table 3 is a comparison among the mammalian Lu-ECAM-1 gene familyshowing approximated nucleic acid similarities (expressed in %).

TABLE 3 bovine murine human human human (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 1) NO: 33) NO: 27) NO: 29) NO: 31) bovine 100 76.7 63.1 85.964.4 (SEQ ID NO: 1) murine — 100 62.6 76.1 61.2 (SEQ ID NO: 33) human —— 100 63.3 58.9 (SEQ ID NO: 27) human — — — 100 62.6 (SEQ ID NO: 29)human — — — — 100 (SEQ ID NO: 31)

EXAMPLE 8

This embodiment illustrates that the full length cDNAs of the presentinvention encode calcium sensitive chloride channels. The various cDNASwere used for transfection of a cell line. For electrophysiologicalstudies, cells were also cotransfected with a reporter vector (pEGFP,CLONTECH). Cotransfection with a reporter vector allows for easyidentification of transfected cells by visualization under a fluorescentmicroscope. Whole cell recording was then carried out in the transfectedcells to determine the presence of calcium sensitive chloride channels.

Transfected cells were used for electrophysiological recording. Cellswere superfused with a bath solution containing 112 mM NMDG-Cl, 30 mMsucrose, 1 mM EGTA, 0.366 mM CaCl₂, 2 mM MgCl₂, 5 mMN-2-hydroxy-xyethanylpiperazine-N-2-ethanesulfonic acid. Whole cellchannel activity was recorded in transfected cells by using borosilicateglass electrodes (tip resistance 4-9 M ohms) filled with the bathsolution. Recordings were carried out in the presence or absence of acalcium channel inhibitors (DIDS, niflumic acid and DTT). To determinethe effect of ionomycin on channel activity, electrodes filled withstandard bath solution containing either 5 mM ATP and 1 mM EGTA in thepresence of low intracellular calcium. After gigaohm seal formation,cells were clamped at +20 mV. Whole cell currents were recorded at roomtemperature, sampled at 5-10 kHz and filtered at 1-2 kHz. The I-Vrelationship was determined using 300 ms voltage steps from a holdingpotential of +20 mV to potentials from −100 to +100 mV at 10 mVintervals. To normalize measured membrane currents to membrane currentsto membrane capacitance, the capacitive current transient recorded inresponse to a 10 mV hyperpolarizing pulse was integrated and divided bythe given voltage to give total membrane capacitance (C_(m)) for eachcell.

As shown in FIG. 9, expression of mCLCA1 in HEK293 cells was associatedwith the appearance of a novel Ca²⁺ sensitive Cl-channel as determinedby whole cell recordings in the presence and absence of the Ca²⁺ionophore ionomycin (2 uM). As shown in FIG. 9b, at low intracellularfree Ca2+ concentrations, the basal current at +100 mV inmCLCA1-transfected cells was 2.05±1.09 pA/pF. With ionomycin the currentincreased to 10.23±3.46 pA/pF. No significant effect of thesemanipulations was seen in non-transfected or control-transfected cells.Basal currents in the presence of 2 mM Ca2+ in transfected cellsaveraged 12.01±6.31 pA/pF. Perfusion of 300 uM DIDS reduced the currentto 1.84±0.96. A similar effect was seen with NFA and DTT. These resultsindicate that the expression of mCLCA1 in HEK293 cells is associatedwith the appearance of a Ca2+ sensitive chloride conductance. Underwhole cell conditions, the current was outwardly rectified and inhibitedby the anion channel blockers DIDS and NFA as well as the reducing agentDTT. This data is summarized in FIG. 10.

Whole cell recording of cells transfected with hCLCA1 cDNA demonstratedthe induction of calcium sensitive chloride channels (FIG. 11). Externalperfusion of ionomycin (2 uM) was associated with an increase in themaximally activated current at +100 mV from 0.65 to 11.06 pA/pF. Thecurrent voltage relationship was outwardly rectified and reversed at 0mV under symmetrical recording conditions. No effect of ionomycin wasobserved on non-transfected cells or control transfected cells. Additionof DIDS, DTT or niflumic acid reduced the currents to 1.63, 1.67 and2.07 pA/pF respectively Cell attached patch recordings of singlechannels confirmed the presence of calcium sensitive anion channel (datanot shown). This data is summarized in FIG. 12.

Whole cell recordings of hCLCA2 transfected HEK293 cells exhibited sslightly outwardly rectifying current/voltage relationship that wasabsent from control cells (transfected with vector alone; FIG. 13). Thiscurrent was sensitive to DIDS (300 uM), DTT (2 mM), niflumic acid (100uM), and tamoxifen (10 uM). When the pipet solution contained low Ca2=(about 25 nM) with 2 mM Ca2+ in the bath, perfusion of the Ca2+ionophore ionomycin (4 uM) through the bath also activated the current(FIG. 13e).

These results indicate that the expression of CACC/AM moleculesdisclosed herein and their variants is associated with the appearance ofcalcium sensitive chloride channels.

EXAMPLE 9

This embodiment illustrates uses of the sequences according to thepresent invention. In one embodiment of the present invention, anindividual having a primary tumor having lung-metastatic capabilities istreated with an anti-adhesion therapy comprising administering to theindividual a therapeutically effective amount of a compositioncomprising either antibody raised to rLu-ECAM-1 or recombinant Lu-ECAM-1complex, or a vector for expressing a soluble form of rLu-ECAM-1 orrLu-ECAM-1 complex which can then bind to the lung-metastatic tumorcells. Either composition may function to prevent lung-metastatic tumorcell adhesion to the lung venule endothelial cells, thereby preventingcolonization by the metastatic tumor cells. As known to those skilled inthe art, an effective amount of a therapeutic composition may depend onthe route of administration (e.g., intravenous or other route known inthe art), and physiological factors including the age, size, and rate ofmetabolism of the individual to be treated.

Another embodiment of the present invention is a method for providingcalcium-dependent chloride conductance channels to mammalian cells.Recombinant Lu-ECAM-1 or rLu-ECAM-1 complex may form a chloride channelwhich may affect chloride secretion, and hence fluid secretion, from thecell. It may be that the chloride ion channel is coupled to the adhesionprocess involving the binding of Lu-ECAM-1 to a ligand, as similarlyobserved for the adherence and growth of lymphatic endothelial cells(Martin et al., 1996, supra). Thus, in mammalian cells in which themembrane chloride ion channels are deficient in number or function(e.g., in airway epithelial cells of cystic fibrosis patients), a methodof providing to mammalian cells a calcium-dependent chloride conductancechannel, rLu-ECAM-1 or rLu-ECAM-1 complex, comprises administeringdirectly to the lung endothelial and/or epithelial cells (in vitro or invivo) an expression vector. The expression vector contains a nucleicacid molecule (or a variant thereof) operably linked to expressioncontrol sequences, wherein the nucleic acid molecule encodes eitherrLu-ECAM-1 or rLu-ECAM-1 complex, with the resultant expression vectorbeing introduced into the mammalian cell, and a functionalcalcium-dependent chloride conductance channel produced in the mammaliancells which contain the expression vector. The cells targeted forchloride cunductance channel production may include airway cellsselected from the group consisting of tracheal, bronchial or lung cells.If the cells are transfected in vitro, the transfected cells may then beintroduced in vivo into the area of the lungs of the individual which isdeficient in chloride channel function.

Having described the preferred embodiments of the present invention, itwill be apparent to one of ordinary skill in the art that variousmodifications may be made to the disclosed embodiments, and that suchmodifications are intended to be within the scope of the presentinvention.

47 1 3317 DNA Unknown sequence encoding Lu-ECAM-1 and Lu-ECAM-1associated protein from bovine endothelial cells 1 ggattccagg gtctccagcattgcctgaat ctggatgtag gtttactgta 50 acatgtgcaa aa atg gtg ctc tgt ctgaat gtt att ctg ttc cta act 98 ttg cat ctc ttg cct gga atg aaa agt tcaatg gta aat ttg att 143 aac aat ggg tat gat ggc att gtc att gca att aacccc agt gtg 188 cca gaa gat gaa aaa ctc att gaa aac ata aag gaa atg gtaact 233 gaa gct tct act tac ctg ttt cat gcc acc aaa cga aga gtt tat 278ttc agg aat gtg agc att tta att cca atg acc tgg aaa tca aaa 323 tct gagtac ttc ata cca aaa caa gaa tca tat gac cag gca gat 368 gtc ata gtt gctaat ccc tat cta aaa tat gga gat gat ccc tat 413 aca ctt caa tat gga aggtgt gga gaa aaa gga aaa tat ata cat 458 ttt act cca aac ttc ttg ttg actaat aat ttc cac atc tat ggg 503 tcc cga ggc aga gta ttt gtc cat gag tgggcc cat ctc cgc tgg 548 gga ata ttt gat gag tat aat gtg gac cag cca ttctat att tcc 593 aga aag aac act att gaa gca aca aga tgt tca act cat attact 638 ggt att aat gtg gtt ttc aag aaa tgc cct gga ggc agc tgt ata 683aca agt cta tgc aga cgt gac tca cag aca ggg ctg tat gaa gca 728 aaa tgtaca ttc ctt cca aaa aaa tcc cag act gca aag gaa tcc 773 att atg ttt atgcca agt ctc cat tct gtg act gaa ttt tgt aca 818 gaa aaa aca cac aat acagaa gct cca aac cta caa aac aaa atg 863 tgc aat ggc aaa agc aca tgg gatgta atc atg aac tct gtt gac 908 ttt cag aat aca tct ccc atg aca gaa atgaat cca ccg act cat 953 cct aca ttt tca ttg ctc aag tcc aaa cag cgg gtagtc tgt ttg 998 gta ctt gat aaa tct gga agc atg tct gca gaa gac cgt ctcttt 1043 caa atg aat caa gca gca gaa cta tac ttg att caa gtt att gaa1088 aag gga tct tta gtt ggg atg gtt aca ttt gac agt gtt gct gaa 1133atc caa aat cat cta aca aga ata act gat gat aat gtt tac caa 1178 aag atcacc gca aaa ctg cct caa gta gct aat ggt gga act tca 1223 att tgt aga gggctc aaa gca gga ttc cag gca att atc cac agt 1268 gac cag agt act tct ggttct gaa atc ata cta tta act gat ggg 1313 gaa gat aat gaa ata aat tca tgcttt gag gat gta aaa cga agt 1358 ggt gca atc atc cac acc att gct ctg ggaccc tct gct gcc aaa 1403 gaa ctg gag aca ttg tca aat atg aca gga gga tatcgt ttt ttt 1448 gcc aat aaa gac ata act ggc ctt act aat gct ttc agt agaatt 1493 tca tct aga agt gga agc atc act cag cag gct att cag ttg gaa1538 agc aaa gcc ttg aaa att aca gga agg aaa aga gta aac ggc aca 1583gtg cct gta gac agt aca gtt gga aat gac act ttc ttt gtt gtc 1628 aca tggaca ata caa aaa cca gaa att gtt ctc caa gat cca aaa 1673 gga aag aaa tataaa acc tcg gat ttc aaa gaa gat aag tta aat 1718 att cga tct gct cgt ctgcaa ata cct ggt att gca gag aca ggt 1763 act tgg act tac agc ctt cta aataat cat gcc agc tct caa atg 1808 cta aca gtg aca gtg acc act cga gca agaagt cct act ata ccc 1853 cca gta att gca aca gct cac atg agt caa cat acagca cat tat 1898 cct agc cca atg att gtt tat gca caa gtc agt caa ggg tttttg 1943 cct gta ctg gga atc agt gta ata gcc att ata gaa acc gaa gat1988 gga cat caa gta aca ttg gag ctc tgg gac aat ggt gca ggt cgt 2033gat act gtc aag aat gat ggc atc tac tca aga tac ttt aca gat 2078 tac tatgga aat ggt aga tac agt tta aaa gta cat gca cag gca 2123 aga aac aac acggct agg cta aat tta aga caa cca cag aac aaa 2168 gtt cta tat gtt cca ggctac gtt gaa aac ggt aaa att ata ctg 2213 aac cca ccc aga cct gaa gtc aaagat gac ctg gca aaa gct aaa 2258 ata gaa gac ttt agc aga cta acc tct ggaggg tca ttt act gta 2303 tca gga gct cct cct cct ggt aat cac cct tct gtgttc cca ccc 2348 agt aaa att aca gat ctt gag gct aag ttc aaa gaa gat tatatt 2393 caa ctt tca tgg aca gcc cct ggc aat gtc cta gat aaa gga aaa2438 gcc aac agc tac att ata aga ata agt aag agt ttc atg gat cgt 2483caa gaa gat ttt gac aat gcg act tta gtg aat act tct aat cta 2528 ata cctaag gag gcc gga tca aaa gaa aat ttt gaa ttt aag cca 2573 gaa cat ttt agagta gaa aat ggc acc aaa ttc tat att tca gtc 2618 caa gcc atc aac gaa gccaat ctc atc tca gag gtt tct cac att 2663 gta caa gca atc aaa ttt att cctcta cca gaa gac agt gtc cat 2708 gat ctg ggt acc aag att tct gaa atc actctg gca att tta gga 2753 tta cca atg att ttc tct gta ttt taaactaggaattgtgtcag 2797 cactgataac caatgttata catagttggt acacatttat ttaggattta2847 attcgctatt ttcttgttct tcagtagcta aattgtgtcc aaccttgcga 2897ctgcaggact gcagcatgcc aggtttccct gtccatcacc aactcccaga 2947 gcttgctcaaatccatgttc atttgagtca gtaatgctaa ctatctcatc 2997 ctctactgcc ctcttctctgtttaccttca atctttcccc agcattagga 3047 tcttttccaa tgagtcagct cttagcatcgggtggccaaa atattggcat 3097 tttcagcaac agttcttcaa atgaaatatc cagggtgattttctttagga 3147 tagactggtg actgacagtt caagggacac tctggagtct tctccagcac3197 cgcaccgcag tttgaaagaa ccagttcttt ggtactcagc cttctttata 3247gtccaatgct cacatctatc atgactcctg gaaaaaccat agctttgaga 3297 aatggatctttgttgggaaa 3317 2 905 PRT Unknown Lu-ECAM-1 precursor from bovineendothelial cells 2 Met Val Leu Cys Leu Asn Val Ile Leu Phe Leu Thr LeuHis Leu -20 -15 -10 Leu Pro Gly Met Lys Ser Ser Met Val Asn Leu Ile AsnAsn Gly -5 1 5 Tyr Asp Gly Ile Val Ile Ala Ile Asn Pro Ser Val Pro GluAsp 10 15 20 Glu Lys Leu Ile Glu Asn Ile Lys Glu Met Val Thr Glu Ala Ser25 30 35 Thr Tyr Leu Phe His Ala Thr Lys Arg Arg Val Tyr Phe Arg Asn 4045 50 Val Ser Ile Leu Ile Pro Met Thr Trp Lys Ser Lys Ser Glu Tyr 55 6065 Phe Ile Pro Lys Gln Glu Ser Tyr Asp Gln Ala Asp Val Ile Val 70 75 80Ala Asn Pro Tyr Leu Lys Tyr Gly Asp Asp Pro Tyr Thr Leu Gln 85 90 95 TyrGly Arg Cys Gly Glu Lys Gly Lys Tyr Ile His Phe Thr Pro 100 105 110 AsnPhe Leu Leu Thr Asn Asn Phe His Ile Tyr Gly Ser Arg Gly 115 120 125 ArgVal Phe Val His Glu Trp Ala His Leu Arg Trp Gly Ile Phe 130 135 140 AspGlu Tyr Asn Val Asp Gln Pro Phe Tyr Ile Ser Arg Lys Asn 145 150 155 ThrIle Glu Ala Thr Arg Cys Ser Thr His Ile Thr Gly Ile Asn 160 165 170 ValVal Phe Lys Lys Cys Pro Gly Gly Ser Cys Ile Thr Ser Leu 175 180 185 CysArg Arg Asp Ser Gln Thr Gly Leu Tyr Glu Ala Lys Cys Thr 190 195 200 PheLeu Pro Lys Lys Ser Gln Thr Ala Lys Glu Ser Ile Met Phe 205 210 215 MetPro Ser Leu His Ser Val Thr Glu Phe Cys Thr Glu Lys Thr 220 225 230 HisAsn Thr Glu Ala Pro Asn Leu Gln Asn Lys Met Cys Asn Gly 235 240 245 LysSer Thr Trp Asp Val Ile Met Asn Ser Val Asp Phe Gln Asn 250 255 260 ThrSer Pro Met Thr Glu Met Asn Pro Pro Thr His Pro Thr Phe 265 270 275 SerLeu Leu Lys Ser Lys Gln Arg Val Val Cys Leu Val Leu Asp 280 285 290 LysSer Gly Ser Met Ser Ala Glu Asp Arg Leu Phe Gln Met Asn 295 300 305 GlnAla Ala Glu Leu Tyr Leu Ile Gln Val Ile Glu Lys Gly Ser 310 315 320 LeuVal Gly Met Val Thr Phe Asp Ser Val Ala Glu Ile Gln Asn 325 330 335 HisLeu Thr Arg Ile Thr Asp Asp Asn Val Tyr Gln Lys Ile Thr 340 345 350 AlaLys Leu Pro Gln Val Ala Asn Gly Gly Thr Ser Ile Cys Arg 355 360 365 GlyLeu Lys Ala Gly Phe Gln Ala Ile Ile His Ser Asp Gln Ser 370 375 380 ThrSer Gly Ser Glu Ile Ile Leu Leu Thr Asp Gly Glu Asp Asn 385 390 395 GluIle Asn Ser Cys Phe Glu Asp Val Lys Arg Ser Gly Ala Ile 400 405 410 IleHis Thr Ile Ala Leu Gly Pro Ser Ala Ala Lys Glu Leu Glu 415 420 425 ThrLys Ser Asn Met Thr Gly Gly Tyr Arg Phe Phe Ala Asn Lys 430 435 440 AspIle Thr Gly Leu Thr Asn Ala Phe Ser Arg Ile Ser Ser Arg 445 450 455 SerGly Ser Ile Thr Gln Gln Ala Ile Gln Leu Glu Ser Lys Ala 460 465 470 LeuLys Ile Thr Gly Arg Lys Arg Val Asn Gly Thr Val Pro Val 475 480 485 AspSer Thr Val Gly Asn Asp Thr Phe Phe Val Val Thr Trp Thr 490 495 500 IleGln Lys Pro Glu Ile Val Leu Gln Asp Pro Lys Gly Lys Lys 505 510 515 TyrLys Thr Ser Asp Phe Lys Glu Asp Lys Leu Asn Ile Arg Ser 520 525 530 AlaArg Leu Gln Ile Pro Gly Ile Ala Glu Thr Gly Thr Trp Thr 535 540 545 TyrSer Leu Leu Asn Asn His Ala Ser Ser Gln Met Leu Thr Val 550 555 560 ThrVal Thr Thr Arg Ala Arg Ser Pro Thr Ile Pro Pro Val Ile 565 570 575 AlaThr Ala His Met Ser Gln His Thr Ala His Tyr Pro Ser Pro 580 585 590 MetIle Val Tyr Ala Gln Val Ser Gln Gly Phe Leu Pro Val Leu 595 600 605 GlyIle Ser Val Ile Ala Ile Ile Glu Thr Glu Asp Gly His Gln 610 615 620 ValThr Leu Glu Leu Trp Asp Asn Gly Ala Gly Arg Asp Thr Val 625 630 635 LysAsn Asp Gly Ile Tyr Ser Arg Tyr Phe Thr Asp Tyr Tyr Gly 640 645 650 AsnGly Arg Tyr Ser Leu Lys Val His Ala Gln Ala Arg Asn Asn 655 660 665 ThrAla Arg Leu Asn Leu Arg Gln Pro Gln Asn Lys Val Leu Tyr 670 675 680 ValPro Gly Tyr Val Glu Asn Gly Lys Ile Ile Leu Asn Pro Pro 685 690 695 ArgPro Glu Val Lys Asp Asp Leu Ala Lys Ala Lys Ile Glu Asp 700 705 710 PheSer Arg Leu Thr Ser Gly Gly Ser Phe Thr Val Ser Gly Ala 715 720 725 ProPro Pro Gly Asn His Pro Ser Val Phe Pro Pro Ser Lys Ile 730 735 740 ThrAsp Leu Glu Ala Lys Phe Lys Glu Asp Tyr Ile Gln Leu Ser 745 750 755 TrpThr Ala Pro Gly Asn Val Leu Asp Lys Gly Lys Ala Asn Ser 760 765 770 TyrIle Ile Arg Ile Ser Lys Ser Phe Met Asp Arg Gln Glu Asp 775 780 785 PheAsp Asn Ala Thr Leu Val Asn Thr Ser Asn Leu Ile Pro Lys 790 795 800 GluAla Gly Ser Lys Glu Asn Phe Glu Phe Lys Pro Glu His Phe 805 810 815 ArgVal Glu Asn Gly Thr Lys Phe Tyr Ile Ser Val Gln Ala Ile 820 825 830 AsnGlu Ala Asn Leu Ile Ser Glu Val Ser His Ile Val Gln Ala 835 840 845 IleLys Phe Ile Pro Leu Pro Glu Asp Ser Val His Asp Leu Gly 850 855 860 ThrLys Ile Ser Glu Ile Thr Leu Ala Ile Leu Gly Leu Pro Met 865 870 875 IlePhe Ser Val Phe 880 884 3 203 PRT Unknown Lu-ECAM-1 associated proteinfrom bovine endothelial cells 3 Val Leu Tyr Val Pro Gly Tyr Val Glu AsnGly Lys Ile Ile Leu 1 5 10 15 Asn Pro Pro Arg Pro Glu Val Lys Asp AspLeu Ala Lys Ala Lys 20 25 30 Ile Glu Asp Phe Ser Arg Leu Thr Ser Gly GlySer Phe Thr Val 35 40 45 Ser Gly Ala Pro Pro Pro Gly Asn His Pro Ser ValPhe Pro Pro 50 55 60 Ser Lys Ile Thr Asp Leu Glu Ala Lys Phe Lys Glu AspTyr Ile 65 70 75 Gln Leu Ser Trp Thr Ala Pro Gly Asn Val Leu Asp Lys GlyLys 80 85 90 Ala Asn Ser Tyr Ile Ile Arg Ile Ser Lys Ser Phe Met Asp Arg95 100 105 Gln Glu Asp Phe Asp Asn Ala Thr Leu Val Asn Thr Ser Asn Leu110 115 120 Ile Pro Lys Glu Ala Gly Ser Lys Glu Asn Phe Glu Phe Lys Pro125 130 135 Glu His Phe Arg Val Glu Asn Gly Thr Lys Phe Tyr Ile Ser Val140 145 150 Gln Ala Ile Asn Glu Ala Asn Leu Ile Ser Glu Val Ser His Ile155 160 165 Val Gln Ala Ile Lys Phe Ile Pro Leu Pro Glu Asp Ser Val His170 175 180 Asp Leu Gly Thr Lys Ile Ser Glu Ile Thr Leu Ala Ile Leu Gly185 190 195 Leu Pro Met Ile Phe Ser Val Phe 200 203 4 26 DNA Artificialsequence Amplification primer 4 aatttaagcc agaacatttt agagta 26 5 23 DNAArtificial sequence Amplification primer 5 gaaaatggca ccaaattcta tat 236 23 DNA Artificial sequence Amplification primer 6 atatagaatttggtgccatt ttc 23 7 19 DNA Artificial sequence Amplification primer 7tagaagtatt cactaaagt 19 8 28 DNA Artificial Sequence Amplificationprimer 8 tactgtctac aggcactgtg ccgtttac 28 9 18 DNA Artificial sequenceAmplification primer 9 ggaatatttg atgagtat 18 10 18 DNA Artificialsequence Amplification primer 10 attcatttga aagagacg 18 11 795 PRTUnknown Variant of Lu-ECAM-1 from bovine endothelial cells 11 Met ValLeu Cys Leu Asn Val Ile Leu Phe Leu Thr Leu His Leu -20 -15 -10 Leu ProGly Met Lys Ser Ser Met Val Asn Leu Ile Asn Asn Gly -5 1 5 Tyr Asp GlyIle Val Ile Ala Ile Asn Pro Ser Val Pro Glu Asp 10 15 20 Glu Lys Leu IleGlu Asn Ile Lys Glu Met Val Thr Glu Ala Ser 25 30 35 Thr Tyr Leu Phe HisAla Thr Lys Arg Arg Val Tyr Phe Arg Asn 40 45 50 Val Ser Ile Leu Ile ProMet Thr Trp Lys Ser Lys Ser Glu Tyr 55 60 65 Phe Ile Pro Lys Gln Glu SerTyr Asp Gln Ala Asp Val Ile Val 70 75 80 Ala Asn Pro Tyr Leu Lys Tyr GlyAsp Asp Pro Tyr Thr Leu Gln 85 90 95 Tyr Gly Arg Cys Gly Glu Lys Gly LysTyr Ile His Phe Thr Pro 100 105 110 Asn Phe Leu Leu Thr Asn Asn Phe HisIle Tyr Gly Ser Arg Gly 115 120 125 Arg Val Phe Val His Glu Trp Ala HisLeu Arg Trp Gly Ile Phe 130 135 140 Asp Glu Tyr Asn Val Asp Gln Pro PheTyr Ile Ser Arg Lys Asn 145 150 155 Thr Ile Glu Ala Thr Arg Cys Ser ThrHis Ile Thr Gly Ile Asn 160 165 170 Val Val Phe Lys Lys Cys Pro Gly GlySer Cys Ile Thr Ser Leu 175 180 185 Cys Arg Arg Asp Ser Gln Thr Gly LeuTyr Glu Ala Lys Cys Thr 190 195 200 Phe Leu Pro Lys Lys Ser Gln Thr AlaLys Glu Ser Ile Met Phe 205 210 215 Met Pro Ser Leu His Ser Val Thr GluPhe Cys Thr Glu Lys Thr 220 225 230 His Asn Thr Glu Ala Pro Asn Leu GlnAsn Lys Met Cys Asn Gly 235 240 245 Lys Ser Thr Trp Asp Val Ile Met AsnSer Val Asp Phe Gln Asn 250 255 260 Thr Ser Pro Met Thr Glu Met Asn ProPro Thr His Pro Thr Phe 265 270 275 Ser Leu Leu Lys Ser Lys Gln Arg ValVal Cys Leu Val Leu Asp 280 285 290 Lys Ser Gly Ser Met Ser Ala Glu AspArg Leu Phe Gln Met Asn 295 300 305 Gln Ala Ala Glu Leu Tyr Leu Ile GlnVal Ile Glu Lys Gly Ser 310 315 320 Leu Val Gly Met Val Thr Phe Asp SerVal Ala Glu Ile Gln Asn 325 330 335 His Leu Thr Arg Ile Thr Asp Asp AsnVal Tyr Gln Lys Ile Thr 340 345 350 Ala Lys Leu Pro Gln Val Ala Asn GlyGly Thr Ser Ile Cys Arg 355 360 365 Gly Leu Lys Ala Gly Phe Gln Ala IleIle His Ser Asp Gln Ser 370 375 380 Thr Ser Gly Ser Glu Ile Ile Leu LeuThr Asp Gly Glu Asp Asn 385 390 395 Glu Ile Asn Ser Cys Phe Glu Asp ValLys Arg Ser Gly Ala Ile 400 405 410 Ile His Thr Ile Ala Leu Gly Pro SerAla Ala Lys Glu Leu Glu 415 420 425 Thr Lys Ser Asn Met Thr Gly Gly TyrArg Phe Phe Ala Asn Lys 430 435 440 Asp Ile Thr Gly Leu Thr Asn Ala PheSer Arg Ile Ser Ser Arg 445 450 455 Ser Gly Ser Ile Thr Gln Gln Ala IleGln Leu Glu Ser Lys Ala 460 465 470 Leu Lys Ile Thr Gly Arg Lys Arg ValAsn Gly Thr Val Pro Val 475 480 485 Asp Ser Thr Val Gly Asn Asp Thr PhePhe Val Val Thr Trp Thr 490 495 500 Ile Gln Lys Pro Glu Ile Val Leu GlnAsp Pro Lys Gly Lys Lys 505 510 515 Tyr Lys Thr Ser Asp Phe Lys Glu AspLys Leu Asn Ile Arg Ser 520 525 530 Ala Arg Leu Gln Ile Pro Gly Ile AlaGlu Thr Gly Thr Trp Thr 535 540 545 Tyr Ser Leu Leu Asn Asn His Ala SerSer Gln Met Leu Thr Val 550 555 560 Thr Val Thr Thr Arg Ala Arg Ser ProThr Ile Pro Pro Val Ile 565 570 575 Ala Thr Ala His Met Ser Gln His ThrAla His Tyr Pro Ser Pro 580 585 590 Met Ile Val Tyr Ala Gln Val Ser GlnGly Phe Leu Pro Val Leu 595 600 605 Gly Ile Ser Val Ile Ala Ile Ile GluThr Glu Asp Gly His Gln 610 615 620 Val Thr Leu Glu Leu Trp Asp Asn GlyAla Gly Arg Asp Thr Val 625 630 635 Lys Asn Asp Gly Ile Tyr Ser Arg TyrPhe Thr Asp Tyr Tyr Gly 640 645 650 Asn Gly Arg Tyr Ser Leu Lys Val HisAla Gln Ala Arg Asn Asn 655 660 665 Thr Ala Arg Leu Asn Leu Arg Gln ProGln Asn Lys Val Leu Tyr 670 675 680 Val Pro Gly Tyr Val Glu Asn Gly LysIle Ile Leu Asn Pro Pro 685 690 695 Arg Pro Glu Val Lys Asp Asp Leu AlaLys Ala Lys Ile Glu Asp 700 705 710 Phe Ser Arg Leu Thr Ser Gly Gly SerPhe Thr Val Ser Gly Ala 715 720 725 Pro Pro Pro Gly Asn His Pro Ser ValPhe Pro Pro Ser Lys Ile 730 735 740 Thr Asp Leu Glu Ala Lys Phe Lys GluAsp Tyr Ile Gln Leu Ser 745 750 755 Trp Thr Ala Pro Gly Asn Val Leu AspLys Gly Lys Ala Glu Ser 760 765 770 774 12 821 PRT Unknown Variant ofLu-ECAM-1 from bovine endothelial cells 12 Met Val Leu Cys Leu Asn ValIle Leu Phe Leu Thr Leu His Leu -20 -15 -10 Leu Pro Gly Met Lys Ser SerMet Val Asn Leu Ile Asn Asn Gly -5 1 5 Tyr Asp Gly Ile Val Ile Ala IleAsn Pro Ser Val Pro Glu Asp 10 15 20 Glu Lys Leu Ile Glu Asn Ile Lys GluMet Val Thr Glu Ala Ser 25 30 35 Thr Tyr Leu Phe His Ala Thr Lys Arg ArgVal Tyr Phe Arg Asn 40 45 50 Val Ser Ile Leu Ile Pro Met Thr Trp Lys SerLys Ser Glu Tyr 55 60 65 Phe Ile Pro Lys Gln Glu Ser Tyr Asp Gln Ala AspVal Ile Val 70 75 80 Ala Asn Pro Tyr Leu Lys Tyr Gly Asp Asp Pro Tyr ThrLeu Gln 85 90 95 Tyr Gly Arg Cys Gly Glu Lys Gly Lys Tyr Ile His Phe ThrPro 100 105 110 Asn Phe Leu Leu Thr Asn Asn Phe His Ile Tyr Gly Ser ArgGly 115 120 125 Arg Val Phe Val His Glu Trp Ala His Leu Arg Trp Gly IlePhe 130 135 140 Asp Glu Tyr Asn Val Asp Gln Pro Phe Tyr Ile Ser Arg LysAsn 145 150 155 Thr Ile Glu Ala Thr Arg Cys Ser Thr His Ile Thr Gly IleAsn 160 165 170 Val Val Phe Lys Lys Cys Pro Gly Gly Ser Cys Ile Thr SerLeu 175 180 185 Cys Arg Arg Asp Ser Gln Thr Gly Leu Tyr Glu Ala Lys CysThr 190 195 200 Phe Leu Pro Lys Lys Ser Gln Thr Ala Lys Glu Ser Ile MetPhe 205 210 215 Met Pro Ser Leu His Ser Val Thr Glu Phe Cys Thr Glu LysThr 220 225 230 His Asn Thr Glu Ala Pro Asn Leu Gln Asn Lys Met Cys AsnGly 235 240 245 Lys Ser Thr Trp Asp Val Ile Met Asn Ser Val Asp Phe GlnAsn 250 255 260 Thr Ser Pro Met Thr Glu Met Asn Pro Pro Thr His Pro ThrPhe 265 270 275 Ser Leu Leu Lys Ser Lys Gln Arg Val Val Cys Leu Val LeuAsp 280 285 290 Lys Ser Gly Ser Met Ser Ala Glu Asp Arg Leu Phe Gln MetAsn 295 300 305 Gln Ala Ala Glu Leu Tyr Leu Ile Gln Val Ile Glu Lys GlySer 310 315 320 Leu Val Gly Met Val Thr Phe Asp Ser Val Ala Glu Ile GlnAsn 325 330 335 His Leu Thr Arg Ile Thr Asp Asp Asn Val Tyr Gln Lys IleThr 340 345 350 Ala Lys Leu Pro Gln Val Ala Asn Gly Gly Thr Ser Ile CysArg 355 360 365 Gly Leu Lys Ala Gly Phe Gln Ala Ile Ile His Ser Asp GlnSer 370 375 380 Thr Ser Gly Ser Glu Ile Ile Leu Leu Thr Asp Gly Glu AspAsn 385 390 395 Glu Ile Asn Ser Cys Phe Glu Asp Val Lys Arg Ser Gly AlaIle 400 405 410 Ile His Thr Ile Ala Leu Gly Pro Ser Ala Ala Lys Glu LeuGlu 415 420 425 Thr Lys Ser Asn Met Thr Gly Gly Tyr Arg Phe Phe Ala AsnLys 430 435 440 Asp Ile Thr Gly Leu Thr Asn Ala Phe Ser Arg Ile Ser SerArg 445 450 455 Ser Gly Ser Ile Thr Gln Gln Ala Ile Gln Leu Glu Ser LysAla 460 465 470 Leu Lys Ile Thr Gly Arg Lys Arg Val Asn Gly Thr Val ProVal 475 480 485 Asp Ser Thr Val Gly Asn Asp Thr Phe Phe Val Val Thr TrpThr 490 495 500 Ile Gln Lys Pro Glu Ile Val Leu Gln Asp Pro Lys Gly LysLys 505 510 515 Tyr Lys Thr Ser Asp Phe Lys Glu Asp Lys Leu Asn Ile ArgSer 520 525 530 Ala Arg Leu Gln Ile Pro Gly Ile Ala Glu Thr Gly Thr TrpThr 535 540 545 Tyr Ser Leu Leu Asn Asn His Ala Ser Ser Gln Met Leu ThrVal 550 555 560 Thr Val Thr Thr Arg Ala Arg Ser Pro Thr Ile Pro Pro ValIle 565 570 575 Ala Thr Ala His Met Ser Gln His Thr Ala His Tyr Pro SerPro 580 585 590 Met Ile Val Tyr Ala Gln Val Ser Gln Gly Phe Leu Pro ValLeu 595 600 605 Gly Ile Ser Val Ile Ala Ile Ile Glu Thr Glu Asp Gly HisGln 610 615 620 Val Thr Leu Glu Leu Trp Asp Asn Gly Ala Gly Arg Asp ThrVal 625 630 635 Lys Asn Asp Gly Ile Tyr Ser Arg Tyr Phe Thr Asp Tyr TyrGly 640 645 650 Asn Gly Arg Tyr Ser Leu Lys Val His Ala Gln Ala Arg AsnAsn 655 660 665 Thr Ala Arg Leu Asn Leu Arg Gln Pro Gln Asn Lys Val LeuTyr 670 675 680 Val Pro Gly Tyr Val Glu Asn Gly Lys Ile Ile Leu Asn ProPro 685 690 695 Arg Pro Glu Val Lys Asp Asp Leu Ala Lys Ala Lys Ile GluAsp 700 705 710 Phe Ser Arg Leu Thr Ser Gly Gly Ser Phe Thr Val Ser GlyAla 715 720 725 Pro Pro Pro Gly Asn His Pro Ser Val Phe Pro Pro Ser LysIle 730 735 740 Thr Asp Leu Glu Ala Lys Phe Lys Glu Asp Tyr Ile Gln LeuSer 745 750 755 Trp Thr Ala Pro Gly Asn Val Leu Asp Lys Gly Lys Ala AlaSer 760 765 770 Gly Ser Phe Pro Met Ser Arg Phe Ser His Gln Val Ala LysVal 775 780 785 Leu Glu Leu Gln Leu Gln His Gln Ser Phe Gln 790 795 80013 342 PRT Unknown Variant of Lu-ECAM-1 from bovine endothelial cells 13Met Val Leu Cys Leu Asn Val Ile Leu Phe Leu Thr Leu His Leu -20 -15 -10Leu Pro Gly Met Lys Ser Ser Met Val Asn Leu Ile Asn Asn Gly -5 1 5 TyrAsp Gly Ile Val Ile Ala Ile Asn Pro Ser Val Pro Glu Asp 10 15 20 Glu LysLeu Ile Glu Asn Ile Lys Glu Met Val Thr Glu Ala Ser 25 30 35 Thr Tyr LeuPhe His Ala Thr Lys Arg Arg Val Tyr Phe Arg Asn 40 45 50 Val Ser Ile LeuIle Pro Met Thr Trp Lys Ser Lys Ser Glu Tyr 55 60 65 Phe Ile Pro Lys GlnGlu Ser Tyr Asp Gln Ala Asp Val Ile Val 70 75 80 Ala Asn Pro Tyr Leu LysTyr Gly Asp Asp Pro Tyr Thr Leu Gln 85 90 95 Tyr Gly Arg Cys Gly Glu LysGly Lys Tyr Ile His Phe Thr Pro 100 105 110 Asn Phe Leu Leu Thr Asn AsnPhe His Ile Tyr Gly Ser Arg Gly 115 120 125 Arg Val Phe Val His Glu TrpAla His Leu Arg Trp Gly Ile Phe 130 135 140 Asp Glu Tyr Asn Val Asp GlnPro Phe Tyr Ile Ser Arg Lys Asn 145 150 155 Thr Ile Glu Ala Thr Arg CysSer Thr His Ile Thr Gly Ile Asn 160 165 170 Val Val Phe Lys Lys Cys ProGly Gly Ser Cys Ile Thr Ser Leu 175 180 185 Cys Arg Arg Asp Ser Gln ThrGly Leu Tyr Glu Ala Lys Cys Thr 190 195 200 Phe Leu Pro Lys Lys Ser GlnThr Ala Lys Glu Ser Ile Met Phe 205 210 215 Met Pro Ser Leu His Ser ValThr Glu Phe Cys Thr Glu Lys Thr 220 225 230 His Asn Thr Glu Ala Pro AsnLeu Gln Asn Lys Met Cys Asn Gly 235 240 245 Lys Ser Thr Trp Asp Val IleMet Asn Ser Val Asp Phe Gln Asn 250 255 260 Thr Ser Pro Met Thr Glu MetAsn Pro Pro Thr His Pro Thr Phe 265 270 275 Ser Leu Leu Lys Ser Lys GlnArg Val Val Cys Leu Val Leu Asp 280 285 290 Lys Ser Gly Ser Met Ser AlaGlu Asp Ile Tyr Leu Leu Ala Leu 295 300 305 Leu Ile Lys Ile Phe Lys LeuIle Gly Asn Thr Ile 310 315 320 321 14 335 DNA Artificial sequenceOligonucleotide probe 14 caacagctac attataagaa taagtaagag tttcatggatcgtcaagaag 50 attttgacaa tgcgacttta gtgaatactt ctaatctaat acctaaggag 100gccggatcaa aagaaaattt tgaatttaag ccagaacatt ttagagtaga 150 aaatggcaccaaattctata tttcagtcca agccatcaac gaagccaatc 200 tcatctcaga ggtttctcacattgtacaag caatcaaatt tattcctcta 250 ccagaagaca gtgtccatga tctgggtaccaagatttctg aaatcactct 300 ggcaatttta ggattaccaa tgattttctc tgtat 335 1517 PRT Artificial sequence Synthetic peptide 15 Glu Asp Glu Lys Leu IleGlu Asn Ile Lys Glu Met Val Thr Glu 5 10 15 Ala Ser 17 16 17 PRTArtificial sequence synthetic peptide 16 Gln Asp Pro Lys Gly Lys Lys TyrLys Thr Ser Asp Phe Lys Glu 1 5 10 15 Asp Lys 17 17 24 DNA ArtificialSequence Amplification primer 17 atgttcaact catattactg gtat 24 18 20 DNAArtificial sequence Amplification primer 18 tgtaggtttg gagcttctgt 20 1920 DNA Artificial Sequence Amplification primer 19 cacagacagg gctgtatgaa20 20 23 DNA Artificial Sequence Amplification Primer 20 ggagatgtattctgaaagtc aac 23 21 24 DNA Artificial Sequence Amplification primer 21atgttcaact catattactg gtac 24 22 20 DNA Artificial SequenceAmplification primer 22 tgtaggtttg gagcttccac 20 23 20 DNA ArtificialSequence Amplification primer 23 cacagacagg gctgtatgag 20 24 23 DNAArtificial Sequence Amplification primer 24 ggagatgtat tttgaaagtc agt 2325 32 DNA Artificial Sequence Amplification primer 25 actgaattcagcagactaac ctctggaggg tc 32 26 32 DNA Artificial Sequence Amplificationprimer 26 tctactagta gctttagcta ctgaagaaca ag 32 27 3007 DNA Homosapiens 27 taacccgcat tttccaaaga gaggaatcac agggagatgt acagca atg ggg 52cca ttt aag agt tct gtg ttc atc ttg att ctt cac ctt cta gaa 97 ggg gccctg agt aat tca ctc att cag ctg aac aac aat ggc tat 142 gaa ggc att gtcgtt gca atc gac ccc aat gtg cca gaa gat gaa 187 aca ctc att caa caa ataaag gac atg gtg acc cag gca tct ctg 232 tat ctg ttt gaa gct aca gga aagcga ttt tat ttc aaa aat gtt 277 gcc att ttg att cct gaa aca tgg aag acaaag gct gac tat gtg 322 aga cca aaa ctt gag acc tac aaa aat gct gat gttctg gtt gct 367 gag tct act cct cca ggt aat gat gaa ccc tac act gag cagatg 412 ggc aac tgt gga gag aag ggt gaa agg atc cac ctc act cct gat 457ttc att gca gga aaa aag tta gct gaa tat gga cca caa ggt aag 502 gca tttgtc cat gag tgg gct cat cta cga tgg gga gta ttt gac 547 gag tac aat aatgat gag aaa ttc tac tta tcc aat gga aga ata 592 caa gca gta aga tgt tcagca ggt att act ggt aca aat gta gta 637 aag aag tgt cag gga ggc agc tgttac acc aaa aga tgc aca ttc 682 aat aaa gtt aca gga ctc tat gaa aaa ggatgt gag ttt gtt ctc 727 caa tcc cgc cag acg gag aag gct tct ata atg tttgca caa cat 772 gtt gat tct ata gtt gaa ttc tgt aca gaa caa aac cac aacaaa 817 gaa gct cca aac aag caa aat caa aaa tgc aat ctc cga agc aca 862tgg gaa gtg atc cgt gat tct gag gac ttt aag aaa acc act cct 907 atg acaaca cag cca cca aat ccc acc ttc tca ttg ctg cag att 952 gga caa aga attgtg tgt tta gtc ctt gac aaa tct gga agc atg 997 gcg act ggt aac cgc ctcaat cga ctg aat caa gca ggc cag ctt 1042 ttc ctg ctg cag aca gtt gag ctgggg tcc tgg gtt ggg atg gtg 1087 aca ttt gac agt gct gcc cat gta caa agtgaa ctc ata cag ata 1132 aac agt ggc agt gac agg gac aca ctc gcc aaa agatta cct gca 1177 gca gct tca gga ggg acg tcc atc tgc agc ggg ctt cga tcggca 1222 ttt act gtg att agg aag aaa tat cca act gat gga tct gaa att1267 gtg ctg ctg acg gat ggg gaa gac aac act ata agt ggg tgc ttt 1312aac gag gtc aaa caa agt ggt gcc atc atc cac aca gtc gct ttg 1357 ggg ccctct gca gct caa gaa cta gag gag ctg tcc aaa atg aca 1402 gga ggt tta cagaca tat gct tca gat caa gtt cag aac aat ggc 1447 ctc att gat gct ttt ggggcc ctt tca tca gga aat gga gct gtc 1492 tct cag cgc tcc atc cag ctt gagagt aag gga tta acc ctc cag 1537 aac agc cag tgg atg aat ggc aca gtg atcgtg gac agc acc gtg 1582 gga aag gac act ttg ttt ctt atc acc tgg aca acgcag cct ccc 1627 caa atc ctt ctc tgg gat ccc agt gga cag aag caa ggt ggcttt 1672 gta gtg gac aaa aac acc aaa atg gcc tac ctc caa atc cca ggc1717 att gct aag gtt ggc act tgg aaa tac agt ctg caa gca agc tca 1762caa acc ttg acc ctg act gtc acg tcc cgt gcg tcc aat gct acc 1807 ctg cctcca att aca gtg act tcc aaa acg aac aag gac acc agc 1852 aaa ttc ccc agccct ctg gta gtt tat gca aat att cgc caa gga 1897 gcc tcc cca att ctc agggcc agt gtc aca gcc ctg att gaa tca 1942 gtg aat gga aaa aca gtt acc ttggaa cta ctg gat aat gga gca 1987 ggt gct gat gct act aag gat gac ggt gtctac tca agg tat ttc 2032 aca act tat gac acg aat ggt aga tac agt gta aaagtg cgg gct 2077 ctg gga gga gtt aac gca gcc aga cgg aga gtg ata ccc cagcag 2122 agt gga gca ctg tac ata cct ggc tgg att gag aat gat gaa ata2167 caa tgg aat cca cca aga cct gaa att aat aag gat gat gtt caa 2212cac aag caa gtg tgt ttc agc aga aca tcc tcg gga ggc tca ttt 2257 gtg gcttct gat gtc cca aat gct ccc ata cct gat ctc ttc cca 2302 cct ggc caa atcacc gac ctg aag gcg gaa att cac ggg ggc agt 2347 ctc att aat ctg act tggaca gct cct ggg gat gat tat gac cat 2392 gga aca gct cac aag tat atc attcga ata agt aca agt att ctt 2437 gat ctc aga gac aag ttc aat gaa tct cttcaa gtg aat act act 2482 gct ctc atc cca aag gaa gcc aac tct gag gaa gtcttt ttg ttt 2527 aaa cca gaa aac att act ttt gaa aat ggc aca gat ctt ttcatt 2572 gct att cag gct gtt gat aag gtc gat ctg aaa tca gaa ata tcc2617 aac att gca cga gta tct ttg ttt att cct cca cag act ccg cca 2662gag aca cct agt cct gat gaa acg tct gct cct tgt cct aat att 2707 cat atcaac agc acc att cct ggc att cac att tta aaa att atg 2752 tgg aag tgg atagga gaa ctg cag ctg tca ata gcc tagggctgaa 2798 tttttgtcag ataaataaaataaatcattc atcctttttt ttgattataa 2848 aattttttaa aatgtatttt agaattcctgtagggggcga tatactaaat 2898 gtatatagta catttatact aaatgtattc ctgtagggggcgatatacta 2948 aatgtatttt agaattcctg tagggggcga taaaataaaa tgctaaacaa2998 ctggggaaa 3007 28 914 PRT Homo sapiens 28 Met Gly Pro Phe Lys SerSer Val Phe Ile Leu Ile Leu His Leu 1 5 10 15 Leu Glu Gly Ala Leu SerAsn Ser Leu Ile Gln Leu Asn Asn Asn 20 25 30 Gly Tyr Glu Gly Ile Val ValAla Ile Asp Pro Asn Val Pro Glu 35 40 45 Asp Glu Thr Leu Ile Gln Gln IleLys Asp Met Val Thr Gln Ala 50 55 60 Ser Leu Tyr Leu Phe Glu Ala Thr GlyLys Arg Phe Tyr Phe Lys 65 70 75 Asn Val Ala Ile Leu Ile Pro Glu Thr TrpLys Thr Lys Ala Asp 80 85 90 Tyr Val Arg Pro Lys Leu Glu Thr Tyr Lys AsnAla Asp Val Leu 95 100 105 Val Ala Glu Ser Thr Pro Pro Gly Asn Asp GluPro Tyr Thr Glu 110 115 120 Gln Met Gly Asn Cys Gly Glu Lys Gly Glu ArgIle His Leu Thr 125 130 135 Pro Asp Phe Ile Ala Gly Lys Lys Leu Ala GluTyr Gly Pro Gln 140 145 150 Gly Lys Ala Phe Val His Glu Trp Ala His LeuArg Trp Gly Val 155 160 165 Phe Asp Glu Tyr Asn Asn Asp Glu Lys Phe TyrLeu Ser Asn Gly 170 175 180 Arg Ile Gln Ala Val Arg Cys Ser Ala Gly IleThr Gly Thr Asn 185 190 195 Val Val Lys Lys Cys Gln Gly Gly Ser Cys TyrThr Lys Arg Cys 200 205 210 Thr Phe Asn Lys Val Thr Gly Leu Tyr Glu LysGly Cys Glu Phe 215 220 225 Val Leu Gln Ser Arg Gln Thr Glu Lys Ala SerIle Met Phe Ala 230 235 240 Gln His Val Asp Ser Ile Val Glu Phe Cys ThrGlu Gln Asn His 245 250 255 Asn Lys Glu Ala Pro Asn Lys Gln Asn Gln LysCys Asn Leu Arg 260 265 270 Ser Thr Trp Glu Val Ile Arg Asp Ser Glu AspPhe Lys Lys Thr 275 280 285 Thr Pro Met Thr Thr Gln Pro Pro Asn Pro ThrPhe Ser Leu Leu 290 295 300 Gln Ile Gly Gln Arg Ile Val Cys Leu Val LeuAsp Lys Ser Gly 305 310 315 Ser Met Ala Thr Gly Asn Arg Leu Asn Arg LeuAsn Gln Ala Gly 320 325 330 Gln Leu Phe Leu Leu Gln Thr Val Glu Leu GlySer Trp Val Gly 335 340 345 Met Val Thr Phe Asp Ser Ala Ala His Val GlnSer Glu Leu Ile 350 355 360 Gln Ile Asn Ser Gly Ser Asp Arg Asp Thr LeuAla Lys Arg Leu 365 370 375 Pro Ala Ala Ala Ser Gly Gly Thr Ser Ile CysSer Gly Leu Arg 380 385 390 Ser Ala Phe Thr Val Ile Arg Lys Lys Tyr ProThr Asp Gly Ser 395 400 405 Glu Ile Val Leu Leu Thr Asp Gly Glu Asp AsnThr Ile Ser Gly 410 415 420 Cys Phe Asn Glu Val Lys Gln Ser Gly Ala IleIle His Thr Val 425 430 435 Ala Leu Gly Pro Ser Ala Ala Gln Glu Leu GluGlu Leu Ser Lys 440 445 450 Met Thr Gly Gly Leu Gln Thr Tyr Ala Ser AspGln Val Gln Asn 455 460 465 Asn Gly Leu Ile Asp Ala Phe Gly Ala Leu SerSer Gly Asn Gly 470 475 480 Ala Val Ser Gln Arg Ser Ile Gln Leu Glu SerLys Gly Leu Thr 485 490 495 Leu Gln Asn Ser Gln Trp Met Asn Gly Thr ValIle Val Asp Ser 500 505 510 Thr Val Gly Lys Asp Thr Leu Phe Leu Ile ThrTrp Thr Thr Gln 515 520 525 Pro Pro Gln Ile Leu Leu Trp Asp Pro Ser GlyGln Lys Gln Gly 530 535 540 Gly Phe Val Val Asp Lys Asn Thr Lys Met AlaTyr Leu Gln Ile 545 550 555 Pro Gly Ile Ala Lys Val Gly Thr Trp Lys TyrSer Leu Gln Ala 560 565 570 Ser Ser Gln Thr Leu Thr Leu Thr Val Thr SerArg Ala Ser Asn 575 580 585 Ala Thr Leu Pro Pro Ile Thr Val Thr Ser LysThr Asn Lys Asp 590 595 600 Thr Ser Lys Phe Pro Ser Pro Leu Val Val TyrAla Asn Ile Arg 605 610 615 Gln Gly Ala Ser Pro Ile Leu Arg Ala Ser ValThr Ala Leu Ile 620 625 630 Glu Ser Val Asn Gly Lys Thr Val Thr Leu GlnLeu Leu Asp Asn 635 640 645 Gly Ala Gly Ala Asp Ala Thr Lys Asp Asp GlyVal Tyr Ser Arg 650 655 660 Tyr Phe Thr Thr Tyr Asp Thr Asn Gly Arg TyrSer Val Lys Val 665 670 675 Arg Ala Leu Gly Gly Val Asn Ala Ala Arg ArgArg Val Ile Pro 680 685 690 Gln Gln Ser Gly Ala Leu Tyr Ile Pro Gly TrpIle Glu Asn Asp 695 700 705 Glu Ile Gln Trp Asn Pro Pro Arg Pro Glu IleAsn Lys Asp Asp 710 715 720 Val Gln His Lys Gln Val Cys Phe Ser Arg ThrSer Ser Gly Gly 725 730 735 Ser Phe Val Ala Ser Asp Val Pro Asn Ala ProIle Pro Asp Leu 740 745 750 Phe Pro Pro Gly Gln Ile Thr Asp Leu Lys AlaGlu Ile His Gly 755 760 765 Gly Ser Leu Ile Asn Leu Thr Trp Thr Ala ProGly Asp Asp Tyr 770 775 780 Asp His Gly Thr Ala His Lys Tyr Ile Ile ArgIle Ser Thr Ser 785 790 795 Ile Leu Asp Leu Arg Asp Lys Phe Asn Glu SerLeu Gln Val Asn 800 805 810 Thr Thr Ala Leu Ile Pro Lys Glu Ala Asn SerGlu Glu Val Phe 815 820 825 Leu Phe Lys Pro Glu Asn Ile Thr Phe Glu AsnGly Thr Asp Leu 830 835 840 Phe Ile Ala Ile Gln Ala Val Asp Lys Val AspLeu Lys Ser Glu 845 850 855 Ile Ser Asn Ile Ala Arg Val Ser Leu Phe IlePro Pro Gln Thr 860 865 870 Pro Pro Glu Thr Pro Ser Pro Asp Glu Thr SerAla Pro Cys Pro 875 880 885 Asn Ile His Ile Asn Ser Thr Ile Pro Gly IleHis Ile Leu Lys 890 895 900 Ile Met Trp Lys Trp Ile Gly Glu Leu Gln LeuSer Ile Ala 905 910 914 29 3418 DNA Homo sapiens 29 tttgtttaac atgcaagaatg gtg ttc agt ctg aag gtg att ctc ttc 48 cta tcc ttg ctt ctc tcg cctgta ttg aaa agc tca ctg gta act 93 ttg aat aac aat gga tat gat ggc attgtg att gca att aat ccc 138 agt gta cca gaa gat gaa aaa ctc att caa aacata aag gaa atg 183 gta act gaa gca tct act cac ctg ttt cat gcc acc aaacaa aga 228 gct tat ttc agg aat gta agc att tta att cca atg acc tac aaa273 tca aaa tct gag tac tta atc cca aaa caa gaa aca tat gac cag 318 gcagat gtc ata gtt gct gat ctt tac ctg aaa tac gga gat gat 363 ccc tat acactt caa tat gga caa tgt gga gat aaa gga caa tat 408 ata cat ttt act ccaaac ttc ttg ttg act aat aac ttg gct acc 453 tat ggg cct cga ggt aaa gtattt gtc cat ggg tgg gcc cat ctc 498 cgg tgg gga gta ttt gat gag tat aatgtg gac cag cca ttc tat 543 att tcc aga aga aac act act gaa gca aca agatgt tcc act cgt 588 att act gtt tac atg gtt ttg aac gaa tgc aag ggg gccagc tgt 633 ata gca cga cca ttc aga cgt gac tca cag aca ggg ctg tat gaa678 gca aaa tgt aca ttt atc cca aag aga tcc cag act gcc aag gaa 723 tccatt gtg ttt atg caa aat ctt gat tct gtg act gaa ttt tgt 768 act gaa aaaaca cac aat aaa gaa gct cca aac cta tat aac aaa 813 atg tgc aat cac agaagc aca tgg gat gta atc atg agc tct gaa 858 gat ttt cag cat tta tct cccatg aca gaa ata aat tta cct cgt 903 cct aca ttt tca ttg ctc aag tcc aaacag cgt gta gtc tgt ttg 948 gta ctt gat aaa tct gga agc atg aat gca gaagac cgt ctc ttt 993 cga atg aat caa gca gca gaa ttg tac ttg att caa attatt gaa 1038 aag gga tcc ttg gtt ggg ttg gtc aca ttt gac agt ttt gct aaa1083 atc caa agt aag ctc ata aaa ata att gat gat aac act tac caa 1128aag atc act gca aac ctg cct caa gaa gct gat ggt ggc act tca 1173 att tgcagg gga ctc aaa gca gga ttt cag gca att ccc cag agt 1218 aat cag agt actttc ggt tct gaa atc ata tta cta aca gat ggg 1263 gaa gat tat caa ata agctta tgc ttt gga gag gta aaa caa agt 1308 ggc aca gtc atc cac acc att gctctg ggg ccg tct gct gac gaa 1353 gaa ctg gag acc ctg tca aat atg aca ggatta cat aag gga cac 1398 tgt tat act gaa agt tca tat agt gct ggg aag ttcatc ttt tgt 1443 gga cat cgt ttt tat gcc cat aaa aac ata aat ggc ctt attgat 1488 gct ttc agc aga att tca tct aga agt ggc agc atc tct cag cag1533 gct ctt cag ttg gaa agt aaa act ttg aat atc cca gcg aag aaa 1578tgg ata aat ggt aca gtg cct gtg gat agt aca gtt aga aat gat 1623 act tccttt gtt gtc aca tgg acg ata caa aag cca gca ata att 1668 ctt caa gat ccaaaa gga aaa aaa tat act acc tca gat ttt caa 1713 gaa ggt gaa cta aat attcgg tct gcc cgt ctt cga ata cca ggt 1758 att gca gag aca ggc act tgg acttac agc gtt cga aac aat cat 1803 acc aaa tct caa ttg cta act gtg aca atgacc act cga gca aga 1848 agc cct acc aca ctc cca gta att gca act gct cacatg agt caa 1893 aat aca gct cat tac cct agc cca gtg att gtt tat gca tgtgtc 1938 agt caa ggg ttt ctt cct gtt ctg gga atc aat gta aca gcc att1983 ata gaa aat gaa gag gga cat caa gta aca ttg gag ctc tgc gac 2028aat ggc gca ggt gct gat tct gtc aag aat gat ggc atc tac tca 2073 agg tatttt aca gat tac cat gga aat ggt aga tac agt tta aaa 2118 gtg ctt acc caggca aga aaa aac aca gct agg cta agt caa caa 2163 cag aat aaa gct ctg tatgta ccg cgc tat gct gaa aat gga aaa 2208 att ata ctg aac cca tcc aaa cctgaa gtc aca gat gat gtg gaa 2253 gga gct caa aca gac gac ttc agc aga ctcacc tct gga ggg tcg 2298 ttt act gta tca gga gtg cct cct aat ggt aat cattct cag gtg 2343 ttc tca cct ggt aaa att gta gac ctc gag gct aag ttt caagga 2388 gat cat att caa ctt tca tgg act gcc cct ggc aag gtc ctc gat2433 aaa gga aga gct gag agc tac att ata aga ata agt aaa cat ttc 2478ctg gac ctc caa gaa gat ttt gat aaa gct gct tta ata aat act 2523 tct ggtctg ata cct aag gag cct ggt tca gta gaa agt ttt gaa 2568 ttt aaa cca gaacct tct aaa ata gag aat ggt acg aca ttc tat 2613 att gca att caa gcc atccat gaa gcc aat gtc acc tca gag gtt 2658 tca aac att gca caa gca act aacttt att cct cca cag gaa ccc 2703 agc att cct gat ctg ggt acc aat att tctgca atc agt ttg gca 2748 att ttt gga tta gct gta att tta tct ata ttt tatact aga aat 2793 tat att aga act caa att caa tgt tat aca tac ttg gta aacatt 2838 tat tta aaa ttt aat tta cta tac tta ttg tct att ata aag ctc2883 att ata ata tat aaa gtg aag tac aaa agt tgt aag ttt cct aat 2928tac ttg att aat tat tac tat ttg agt tat tat atg tta atc aaa 2973 atg agtata tca ttt cct gtc tgg aat aat cca ctc att aat ttt 3018 taatatgaaaagatatatat ttgtacttgt aagcatttta agaaacattt 3068 ttaaagtgtg ctacaaattcatttggtgta ctaacatcaa aatgtatcca 3118 agccatttaa aaaatattta tatatacatagtagcaaata gttttataga 3168 tttatttgta tcgcattttt tattacaaat gaatatttcatgtttatata 3218 agctgtaatc aaaaaggact agtagtagta gtaaggaagt caaatttgtt3268 tttttatcat tgattataag tggtatattt gttttttgtc attgattaaa 3318agtgatttta gccctaggcc cgaaatgact agcaaatatc attttctgta 3368 tgaattgtggaacatcacaa taaaattatt tctgtgctga tgctaaaaaa 3418 30 1000 PRT Homosapiens 30 Met Val Phe Ser Leu Lys Val Ile Leu Phe Leu Ser Leu Leu Leu 15 10 15 Ser Pro Val Leu Lys Ser Ser Leu Val Thr Leu Asn Asn Asn Gly 2025 30 Tyr Asp Gly Ile Val Ile Ala Ile Asn Pro Ser Val Pro Glu Asp 35 4045 Glu Lys Leu Ile Gln Asn Ile Lys Glu Met Val Thr Gln Ala Ser 50 55 60Thr His Leu Phe His Ala Thr Lys Gln Arg Ala Tyr Phe Arg Asn 65 70 75 ValSer Ile Leu Ile Pro Met Thr Tyr Lys Ser Lys Ser Glu Tyr 80 85 90 Leu IlePro Lys Gln Glu Thr Tyr Asp Gln Ala Asp Val Ile Val 95 100 105 Ala AspLeu Tyr Leu Lys Tyr Gly Asp Asp Pro Tyr Thr Leu Gln 110 115 120 Tyr GlyGln Cys Gly Asp Lys Gly Gln Tyr Ile His Phe Thr Pro 125 130 135 Asn PheLeu Leu Thr Asn Asn Leu Ala Thr Tyr Gly Pro Arg Gly 140 145 150 Lys ValPhe Val His Gly Trp Ala His Leu Arg Trp Gly Val Phe 155 160 165 Asp GluTyr Asn Val Asp Gln Pro Phe Tyr Ile Ser Arg Arg Asn 170 175 180 Thr ThrGlu Ala Thr Arg Cys Ser Thr Arg Ile Thr Val Tyr Met 185 190 195 Val LeuAsn Glu Cys Lys Gly Ala Ser Cys Ile Ala Arg Pro Phe 200 205 210 Arg ArgAsp Ser Gln Thr Gly Leu Tyr Glu Ala Lys Cys Thr Phe 215 220 225 Ile ProLys Arg Ser Gln Thr Ala Lys Glu Ser Ile Val Phe Met 230 235 240 Gln AsnLeu Asp Ser Val Thr Glu Phe Cys Thr Glu Lys Thr His 245 250 255 Asn LysGlu Ala Pro Asn Leu Tyr Asn Lys Met Cys Asn His Arg 260 265 270 Ser ThrTrp Asp Val Ile Met Ser Ser Glu Asp Phe Gln His Leu 275 280 285 Ser ProMet Thr Glu Ile Asn Leu Pro Arg Pro Thr Phe Ser Leu 290 295 300 Leu LysSer Lys Gln Arg Val Val Cys Leu Val Leu Asp Lys Ser 305 310 315 Gly SerMet Asn Ala Glu Asp Arg Leu Phe Arg Met Asn Gln Ala 320 325 330 Ala GluLeu Tyr Leu Ile Gln Ile Ile Glu Lys Gly Ser Leu Val 335 340 345 Gly LeuVal Thr Phe Asp Ser Phe Ala Lys Ile Gln Ser Lys Leu 350 355 360 Ile LysIle Ile Asp Asp Asn Thr Tyr Gln Lys Ile Thr Ala Asn 365 370 375 Leu ProGln Glu Ala Asp Gly Gly Thr Ser Ile Cys Arg Gly Leu 380 385 390 Lys AlaGly Phe Gln Ala Ile Pro Gln Ser Asn Gln Ser Thr Phe 395 400 405 Gly SerGlu Ile Ile Leu Leu Thr Asp Gly Glu Asp Tyr Gln Ile 410 415 420 Ser LeuCys Phe Gly Glu Val Lys Gln Ser Gly Thr Val Ile His 425 430 435 Thr IleAla Leu Gly Pro Ser Ala Asp Glu Glu Leu Glu Thr Leu 440 445 450 Ser AsnMet Thr Gly Leu His Lys Gly His Cys Tyr Thr Glu Ser 455 460 465 Ser TyrSer Ala Gly Lys Phe Ile Phe Cys Gly His Arg Phe Tyr 470 475 480 Ala HisLys Asn Ile Asn Gly Leu Ile Asp Ala Phe Ser Arg Ile 485 490 495 Ser SerArg Ser Gly Ser Ile Ser Gln Gln Ala Leu Gln Leu Glu 500 505 510 Ser LysThr Leu Asn Ile Pro Ala Lys Lys Trp Ile Asn Gly Thr 515 520 525 Val ProVal Asp Ser Thr Val Arg Asn Asp Thr Ser Phe Val Val 530 535 540 Thr TrpThr Ile Gln Lys Pro Ala Ile Ile Leu Gln Asp Pro Lys 545 550 555 Gly LysLys Tyr Thr Thr Ser Asp Phe Gln Glu Gly Glu Leu Asn 560 565 570 Ile ArgSer Ala Arg Leu Arg Ile Pro Gly Ile Ala Glu Thr Gly 575 580 585 Ile TrpThr Tyr Ser Val Arg Asn Asn His Thr Lys Ser Gln Leu 590 595 600 Leu ThrVal Thr Met Thr Thr Arg Ala Arg Ser Pro Thr Thr Leu 605 610 615 Pro ValIle Ala Thr Ala His Ser Met Gln Asn Thr Ala His Tyr 620 625 630 Pro SerPro Val Ile Val Tyr Ala Cys Val Ser Gln Gly Phe Leu 635 640 645 Pro ValLeu Gly Ile Asn Val Thr Ala Ile Ile Glu Asn Glu Glu 650 655 660 Gly HisGln Val Thr Leu Glu Leu Cys Asp Asn Gly Ala Gly Ala 665 670 675 Asp SerVal Lys Asn Asp Gly Ile Tyr Ser Arg Tyr Phe Thr Asp 680 685 690 Tyr HisGly Asn Gly Arg Tyr Ser Leu Lys Val Leu Thr Gln Ala 695 700 705 Arg LysAsn Thr Ala Arg Leu Ser Gln Gln Gln Asn Lys Ala Leu 710 715 720 Tyr ValPro Arg Tyr Ala Glu Asn Gly Lys Ile Ile Leu Asn Pro 725 730 735 Ser LysPro Glu Val Thr Asp Asp Val Glu Gly Ala Gln Thr Asp 740 745 750 Asp PheSer Arg Leu Thr Ser Gly Gly Ser Phe Thr Val Ser Gly 755 760 765 Val ProPro Asn Gly Asn His Ser Gln Val Phe Ser Pro Gly Lys 770 775 780 Ile ValAsp Leu Glu Ala Lys Phe Gln Gly Asp His Ile Gln Leu 785 790 795 Ser TrpThr Ala Pro Gly Lys Val Leu Asp Lys Gly Arg Ala Glu 800 805 810 Ser TyrIle Ile Arg Ile Ser Lys His Phe Leu Asp Leu Gln Glu 815 820 825 Asp PheAsp Lys Ala Ala Leu Ile Asn Thr Ser Gly Leu Ile Pro 830 835 840 Lys GluPro Gly Ser Val Glu Ser Phe Glu Phe Lys Pro Glu Pro 845 850 855 Ser LysIle Glu Asn Gly Thr Thr Phe Tyr Ile Ala Ile Gln Ala 860 865 870 Ile HisGlu Ala Asn Val Thr Ser Glu Val Ser Asn Ile Ala Gln 875 880 885 Ala ThrAsn Phe Ile Pro Pro Gln Glu Pro Ser Ile Pro Asp Leu 890 895 900 Gly ThrAsn Ile Ser Ala Ile Ser Leu Ala Ile Phe Gly Leu Ala 905 910 915 Val IleLeu Ser Ile Phe Tyr Thr Arg Asn Tyr Ile Arg Thr Gln 920 925 930 Ile GlnCys Tyr Thr Tyr Leu Val Asn Ile Tyr Leu Lys Phe Asn 935 940 945 Leu LeuTyr Leu Leu Ser Ile Ile Lys Leu Ile Ile Ile Tyr Lys 950 955 960 Val LysTyr Lys Ser Cys Lys Phe Pro Asn Tyr Leu Ile Asn Tyr 965 970 975 Tyr TyrLeu Ser Tyr Tyr Met Leu Ile Lys Met Ser Ile Ser Phe 980 985 990 Pro ValTrp Asn Asn Pro Leu Ile Asn Phe 995 1000 31 2970 DNA Homo sapiens 31acctaaaacc ttgcaagttc aggaagaaac catctgcatc catattgaaa 50 acctgacacaatgtatgcag caggctcagt gtgagtgaac tggaggcttc 100 tctacaac atg acc caa aggagc att gca ggt cct att tgc aac 144 ctg aag ttt gtg act ctc ctg gtt gcctta agt tca gaa ctc cca 189 ttc ctg gga gct gga gta cag ctt caa gac aatggg tat aat gga 234 ttg ctc att gca att aat cct cag gta cct gag aat cagaac ctc 279 atc tca aac att aag gaa atg ata act gaa gct tca ttt tac cta324 ttt aat gct acc aag aga aga gta ttt ttc aga aat ata aag att 369 ttaata cct gcc aca tgg aaa gct aat aat aac agc aaa ata aaa 414 caa gaa tcatat gaa aag gca aat gtc ata gtg act gac tgg tat 459 ggg gca cat gga gatgat cca tac acc cta caa tac aga ggg tgt 504 gga aaa gag gga aaa tac attcat ttc aca cct aat ttc cta ctg 549 aat gat aac tta aca gct ggc tac ggatca cga ggc cga gtg ttt 594 gtc cat gaa tgg gcc cac ctc cgt tgg ggt gtgttc gat gag tat 639 aac aat gac aaa cct ttc tac ata aat ggg caa aat caaatt aaa 684 gtg aca agg tgt tca tct gac atc aca ggc att ttt gtg tgt gaa729 aaa ggt cct tgc ccc caa gaa aac tgt att att agt aag ctt ttt 774 aaagaa gga tgc acc ttt atc tac aat agc acc caa aat gca act 819 gca tca ataatg ttc atg caa agt tta tct tct gtg gtt gaa ttt 864 tgt aat gca agt acccac aac caa gaa gca cca aac cta cag aac 909 cag atg tgc agc ctc aga agtgca tgg gat gta atc aca gac tct 954 gct gac ttt cac cac agc ttt ccc atgaat ggg act gag ctt cca 999 cct cct ccc aca ttc tcg ctt gta cag gct ggtgac aaa gtg gtc 1044 tgt tta gtg ctg gat gtg tcc agc aag atg gca gag gctgac aga 1089 ctc ctt caa cta caa caa gcc gca gaa ttt tat ttg atg cag att1134 gtt gaa att cat acc ttc gtg ggc att gcc agt ttc gac agc aaa 1179gga gag atc aga gcc cag cta cac caa att aac agc aat gat gat 1224 cga aagttg ctg gtt tca tat ctg ccc acc act gta tca gct aaa 1269 aca gac atc agcatt tgt tca ggg ctt aag aaa gga ttt gag gtg 1314 gtt gaa aaa ctg aat ggaaaa gct tat ggc tct gtg atg ata tta 1359 gtg acc agc gga gat gat aag cttctt ggc aat tgc tta ccc act 1404 gtg ctc agc agt ggt tca aca att cac tccatt gcc ctg ggt tca 1449 tct gca gcc cca aat ctg gag gaa tta tca cgt cttaca gga ggt 1494 tta aag ttc ttt gtt cca gat ata tca aac tcc aat agc atgatt 1539 gat gct ttc agt aga att tcc tct gga act gga gac att ttc cag1584 caa cat att cag ctt gaa agt aca ggt gaa aat gtc aaa cct cac 1629cat caa ttg aaa aac aca gtg act gtg gat aat act gtg ggc aac 1674 gac actatg ttt cta gtt acg tgg cag gcc agt ggt cct cct gag 1719 att ata tta tttgat cct gat gga cga aaa tac tac aca aat aat 1764 ttt atc acc aat cta actttt cgg aca gct agt ctt tgg att cca 1809 gga aca gct aag cct ggg cac tggact tac acc ctg aac aat acc 1854 cat cat tct ctg caa gcc ctg aaa gtg acagtg acc tct cgc gcc 1899 tcc aac tca gct gtg ccc cca gcc act gtg gaa gccttt gtg gaa 1944 aga gac agc ctc cat ttt cct cat cct gtg atg att tat gccaat 1989 gtg aaa cag gga ttt tat ccc att ctt aat gcc act gtc act gcc2034 aca gtt gag cca gag act gga gat cct gtt acg ctg aga ctc ctt 2079gat gat gga gca ggt gct gat gtt ata aaa aat gat gga att tac 2124 tcg aggtat ttt ttc tcc ttt gct gca aat ggt aga tat agc ttg 2169 aaa gtg cat gtcaat cac tct ccc agc ata agc acc cca gcc cac 2214 tct att cca ggg agt catgct atg tat gta cca ggt tac aca gca 2259 aac ggt aat att cag atg aat gctcca agg aaa tca gta ggc aga 2304 aat gag gag gag cga aag tgg ggc ttt agccga gtc agc tca gga 2349 ggc tcc ttt tca gtg ctg gga gtt cca gct ggc ccccac cct gat 2394 gtg ttt cca cca tgc aaa att att gac ctg gaa gct gta aaagta 2439 gaa gag gaa ttg acc cta tct tgg aca gca cct gga gaa gac ttt2484 gat cag ggc cag gct aca agc tat gaa ata aga atg agt aaa agt 2529cta cag aat atc caa gat gac ttt aac aat gct att tta gta aat 2574 aca tcaaag cga aat cct cag caa gct ggc atc agg gag ata ttt 2619 acg ttc tca ccccag att tcc acg aat gga cct gaa cat cag cca 2664 aat gga gaa aca cat gaaagc cac aga att tat gtt gca ata cga 2709 gca atg gat agg aac tcc tta cagtct gct gta tct aac att gcc 2754 cag gcg cct ctg ttt att ccc ccc aat tctgat cct gta cct gcc 2799 aga gat tat ctt ata ttg aaa gga gtt tta aca gcaatg ggt ttg 2844 ata gga atc att tgc ctt att ata gtt gtg aca cat cat acttta 2889 agc agg aaa aag aga gca gac aag aaa gag aat gga aca aaa tta2934 tta taaataaata tccaaagtgt cttccttctc aaa 2970 32 943 PRT Homosapiens 32 Met Thr Gln Arg Ser Ile Ala Gly Pro Ile Cys Asn Leu Lys Phe 15 10 15 Val Thr Leu Leu Val Ala Leu Ser Ser Glu Leu Pro Phe Leu Gly 2025 30 Ala Gly Val Gln Leu Gln Asp Asn Gly Tyr Asn Gly Leu Leu Ile 35 4045 Ala Ile Asn Pro Gln Val Pro Glu Asn Gln Asn Leu Ile Ser Asn 50 55 60Ile Lys Glu Met Ile Thr Glu Ala Ser Phe Tyr Leu Phe Asn Ala 65 70 75 ThrLys Arg Arg Val Phe Phe Arg Asn Ile Lys Ile Leu Ile Pro 80 85 90 Ala ThrTrp Lys Ala Asn Asn Asn Ser Lys Ile Lys Gln Glu Ser 95 100 105 Tyr GluLys Ala Asn Val Ile Val Thr Asp Trp Tyr Gly Ala His 110 115 120 Gly AspAsp Pro Tyr Thr Leu Gln Tyr Arg Gly Cys Gly Lys Glu 125 130 135 Gly LysTyr Ile His Phe Thr Pro Asn Phe Leu Leu Asn Asp Asn 140 145 150 Leu ThrAla Gly Tyr Gly Ser Arg Gly Arg Val Phe Val His Glu 155 160 165 Trp AlaHis Leu Arg Trp Gly Val Phe Asp Glu Tyr Asn Asn Asp 170 175 180 Lys ProPhe Tyr Ile Asn Gly Gln Asn Gln Ile Lys Val Thr Arg 185 190 195 Cys SerSer Asp Ile Thr Gly Ile Phe Val Cys Glu Lys Gly Pro 200 205 210 Cys ProGln Glu Asn Cys Ile Ile Ser Lys Leu Phe Lys Glu Gly 215 220 225 Cys ThrPhe Ile Tyr Asn Ser Thr Gln Asn Ala Thr Ala Ser Ile 230 235 240 Met PheMet Gln Ser Leu Ser Ser Val Val Glu Phe Cys Asn Ala 245 250 255 Ser ThrHis Asn Gln Glu Ala Pro Asn Leu Gln Asn Gln Met Cys 260 265 270 Ser LeuArg Ser Ala Trp Asp Val Ile Thr Asp Ser Ala Asp Phe 275 280 285 His HisSer Phe Pro Met Asn Gly Thr Glu Leu Pro Pro Pro Pro 290 295 300 Thr PheSer Leu Val Gln Ala Gly Asp Lys Val Val Cys Leu Val 305 310 315 Leu AspVal Ser Ser Lys Met Ala Glu Ala Asp Arg Leu Leu Gln 320 325 330 Leu GlnGln Ala Ala Glu Phe Tyr Leu Met Gln Ile Val Glu Ile 335 340 345 His ThrPhe Val Gly Ile Ala Ser Phe Asp Ser Lys Gly Glu Ile 350 355 360 Arg AlaGln Leu His Gln Ile Asn Ser Asn Asp Asp Arg Lys Leu 365 370 375 Leu ValSer Tyr Leu Pro Thr Thr Val Ser Ala Lys Thr Asp Ile 380 385 390 Ser IleCys Ser Gly Leu Lys Lys Gly Phe Glu Val Val Glu Lys 395 400 405 Leu AsnGly Lys Ala Tyr Gly Ser Val Met Ile Leu Val Thr Ser 410 415 420 Gly AspAsp Lys Leu Leu Gly Asn Cys Leu Pro Thr Val Leu Ser 425 430 435 Ser GlySer Thr Ile His Ser Ile Ala Leu Gly Ser Ser Ala Ala 440 445 450 Pro AsnLeu Glu Glu Leu Ser Arg Leu Thr Gly Gly Leu Lys Phe 455 460 465 Phe ValPro Asp Ile Ser Asn Ser Asn Ser Met Ile Asp Ala Phe 470 475 480 Ser ArgIle Ser Ser Gly Thr Gly Asp Ile Phe Gln Gln His Ile 485 490 495 Gln LeuGlu Ser Thr Gly Glu Asn Val Lys Pro His His Gln Leu 500 505 510 Lys AsnThr Val Thr Val Asp Asn Thr Val Gly Asn Asp Ile Met 515 520 525 Phe LeuVal Thr Trp Gln Ala Ser Gly Pro Pro Glu Ile Ile Leu 530 535 540 Phe AspPro Asp Gly Arg Lys Tyr Tyr Thr Asn Asn Phe Thr Thr 545 550 555 Asn LeuThr Phe Arg Thr Ala Ser Leu Trp Ile Pro Gly Thr Ala 560 565 570 Lys ProGly His Trp Thr Tyr Thr Leu Asn Asn Thr His His Ser 575 580 585 Leu GlnAla Leu Lys Val Thr Val Thr Ser Arg Ala Ser Asn Ser 590 595 600 Ala ValPro Pro Ala Thr Val Glu Ala Phe Val Glu Arg Asp Ser 605 610 615 Leu HisPhe Pro His Pro Val Met Ile Tyr Ala Asn Val Lys Gln 620 625 630 Gly PheTyr Pro Ile Ile Asn Ala Thr Val Thr Ala Thr Val Glu 635 640 645 Pro GluThr Gly Asp Pro Val Thr Leu Arg Leu Leu Asp Asp Gly 650 655 660 Ala GlyAla Asp Val Ile Lys Asn Asp Gly Ile Tyr Ser Arg Tyr 665 670 675 Phe PheSer Phe Ala Ala Asn Gly Arg Tyr Ser Leu Lys Val His 680 685 690 Val AsnHis Ser Pro Ser Ile Ser Thr Pro Ala His Ser Ile Pro 695 700 705 Gly SerHis Ala Met Tyr Val Pro Gly Tyr Thr Ala Asn Gly Asn 710 715 720 Ile GlnMet Asn Ala Pro Arg Lys Ser Val Gly Arg Asn Glu Glu 725 730 735 Glu ArgLys Trp Gly Phe Ser Arg Val Ser Ser Gly Gly Ser Phe 740 745 750 Ser ValLeu Gly Val Pro Ala Gly Pro His Pro Asp Val Phe Pro 755 760 765 Pro CysLys Ile Ile Asp Leu Glu Ala Val Lys Val Glu Glu Glu 770 775 780 Leu ThrLeu Ser Trp Thr Ala Pro Gly Glu Asp Phe Asp Gln Gly 785 790 795 Gln AlaThr Ser Tyr Glu Ile Arg Met Ser Lys Ser Leu Gln Asn 800 805 810 Ile GlnAsp Asp Phe Asn Asn Ala Ile Leu Val Asn Thr Ser Lys 815 820 825 Arg AsnPro Gln Gln Ala Gly Ile Arg Glu Ile Phe Thr Phe Ser 830 835 840 Pro GlnIle Ser Thr Asn Gly Pro Glu His Gln Pro Asn Gly Glu 845 850 855 Thr HisGlu Ser His Arg Ile Tyr Val Ala Ile Arg Ala Met Asp 860 865 870 Arg AsnSer Leu Gln Ser Ala Val Ser Asn Ile Ala Gln Ala Pro 875 880 885 Leu PheIle Pro Pro Asn Ser Asp Pro Val Pro Ala Arg Asp Tyr 890 895 900 Leu IleLeu Lys Gly Val Leu Thr Ala Met Gly Leu Ile Gly Ile 905 910 915 Ile CysLeu Ile Ile Val Val Thr His His Thr Leu Ser Arg Lys 920 925 930 Lys ArgAla Asp Lys Lys Glu Asn Gly Thr Lys Leu Leu 935 940 943 33 3022 DNA Musmusculus 33 actggagcag tgcgacc atg gtg cca ggg ctg cag gtc ctt ctg ttc47 ctc acc ctg cat ctc ctg cag aac aca gag agc tcc atg gtg cat 92 ctcaac agc aat gga tac gag ggt gtg gtc att gcc att aac ccc 137 agt gtg ccagag gac gaa agg ctc atc cca agc ata aag gaa atg 182 gta act caa gct tctacc tac ctg ttt gaa gcc agc caa gga aga 227 gtt tat ttc agg aac ata agcata tta gtc ccg atg acc tgg aag 272 tcg aaa tct gag tac tta atg cca aaacga gaa tcg tac gac aaa 317 gca gac gtc ata gtt gcg gat cct cac ctg caacat gga gac gac 362 ccc tac acc ctt cag tat gga cag tgt ggg gac aga ggacag tac 407 ata cac ttc act cca aac ttc cta ctc act gat aac ttg cgt atc452 tat gga ccc cga ggc aga gtc ttt gtc cat gag tgg gcc cat ctc 497 cggtgg gga gta ttt gat gag tat aac gtg gac cgg tca ctt tac 542 att tct agaaag aac act ata gaa gca aca agg tgc tcc gcc agc 587 atc aca ggc aag aaggtg gtc cac gag tgt cag aga ggc agc tgt 632 gtg aca agg gcg tgc cgg cgtgac tcg aag aca cgg ctg tat gaa 677 ccc aaa tgt aca ttt atc cca gac aaaata cag aca gct ggg gcc 722 tcc ata atg ttc atg caa aac ctc aat tct gtggtt gaa ttt tgc 767 aca gaa aat aac cac aat gca gaa gcc cca aac cta caaaac aaa 812 atg tgc aat cgc aga agc acg tgg gat gta atc aag acg tct gct857 gac ttt cag aat gcc cct ccc atg aga gga aca gaa gcc cct cct 902 ccacct aca ttt tat ctg ctc aag tcc aga agg cga gtg gtg tgc 947 ttg gtg ctggat aaa tct gga agc atg gac aaa gaa gac cgt ctt 992 att cga atg aat caagca gca gaa ctg tac tta act caa att gtg 1037 gaa aag gag tct atg gtt ggatta gtc aca ttt gac agc gct gcc 1082 cac atc caa aat tat cta ata aaa ataacg agt agt agt gac tac 1127 caa aag atc acc gca aac ctc ccc caa cag gcttct ggt gga act 1172 tca att tgc cat gga ctc cag gca gga ttt cag gca attacc tcc 1217 agt gac cag agc act tcc ggt tct gag atc gta ttg ctg aca gat1262 ggg gaa gat aat gga ata cgt tcc tgc ttt gag gcc gtc tct cgc 1307agc ggt gcc atc atc cac acc atc gct ctg ggg cct tcg cgt gcc 1352 cga gaactg gag act ctg tcg gac atg aca gga ggg ctt cgt ttc 1397 tat gcc aac aaagac cta aac agc ctt atc gat gct ttc agt aga 1442 att tca tct aca agt ggcagc gtc tcc cag cag gct ctg cag ttg 1487 gag agc aaa gcc ttc gat gtc agagca ggg gca tgg ata aac ggt 1532 aca gta cct ctg gac agt acc gtc ggc aacgac acg ttc ttt gtt 1577 atc acc tgg atg gta aaa aag cca gaa atc att cttcaa gat cca 1622 aaa gga aaa aaa tat aca acc tca gat ttc caa gat gat aaacta 1667 aac atc cgg tct gct aga ctt caa ata ccg ggc act gca gag aca1712 ggt act tgg act tac agc tac acg ggt acc aag tct cag ttg att 1757aca atg aca gtg acc act cga gca aga agt ccc acc atg gaa cca 1802 ctc ctgggc tac tgc tac atg agt cag agc aca gcc cag tac cct 1847 agc cgg atg attgtg tac gca cgg gtc agc caa gga ttt ttg cct 1892 gtt ctg gga gcc aat gtcaca gcc ctc ata gaa gct gaa cat gga 1937 cat caa gtc acc ttg gag ctc tgggac aat ggg gca ggt gct gat 1982 atc gtt aaa aat gat ggc atc tac aca agatac ttt aca gat tat 2027 cat gga aat ggt aga tac agc cta aaa gtg cgt gtccag gca caa 2072 aga aac aaa acc aga ctg agc tta aga cag aag aac aag tcttta 2117 tat ata cct ggc tat gtg gaa aat ggt aaa att gta ctg aat cca2162 ccc aga cca gat gtc caa gaa gaa gcc ata gaa gct aca gtg gaa 2207gac ttc aac aga gta acc tct gga ggg tcg ttt act gtg tct gga 2252 gcg ccccct gat ggc gac cac gct cgt gtg ttc cca cca agt aaa 2297 gtc aca gac ctggag gct gag ttt ata ggt gat tat att cac ctt 2342 aca tgg acg gcc cct ggcaag gtt ctc gac aat gga aga gca cat 2387 aga tac atc atc aga atg agc cagcat cct ctg gat ctc caa gaa 2432 gat ttt aac aat gct act tta gtg aat gcttcc agt ctg ata cct 2477 aaa gaa gct ggc tca aaa gaa gca ttt aaa ttc aaacca gaa act 2522 ttt aaa ata gca aat ggc atc cag ctc tac att gca atc caggca 2567 gac aat gaa gcc agt ctc acc tct gag gtc tcc aac atc gca cag2612 gct gtc aag ctt act tct cta gaa gat agt atc tct gca ctg ggt 2657gat gat att tct gca atc tct atg aca att tgg ggg tta act gtg 2702 att tttaac tct att tta aac tagaagatag aatggcacta 2743 aaatgcaatc ctgtacatatttgctaagtg ttgctttaga atgtctttac 2793 tacacactca aaggctgcct gtcaacaattgtaatataga agttcatatt 2843 caaagttgaa aatcccgagt tactaacaca attcttttgctatatgtaga 2893 tcaagattaa cagttcctca ttcaatttct taattgttcc atttactatg2943 gaaataagat atccattctc ttttcacagt gtgatgcaag ttcactttgt 2993atatgaaaat aaaaaatttg tacaactcg 3022 34 902 PRT Mus musculus 34 Met ValPro Gly Leu Gln Val Leu Leu Phe Leu Thr Leu His Leu 5 10 15 Leu Gln AsnThr Glu Ser Ser Met Val His Leu Asn Ser Asn Gly 20 25 30 Tyr Glu Gly ValVal Ile Ala Ile Asn Pro Ser Val Pro Glu Asp 35 40 45 Glu Arg Leu Ile ProSer Ile Lys Glu Met Val Thr Gln Ala Ser 50 55 60 Thr Tyr Leu Phe Glu AlaSer Gln Gly Arg Val Tyr Phe Arg Asn 65 70 75 Ile Ser Ile Leu Val Pro MetThr Trp Lys Ser Lys Ser Glu Tyr 80 85 90 Leu Met Pro Lys Arg Glu Ser TyrAsp Lys Ala Asp Val Ile Val 95 100 105 Ala Asp Pro His Leu Gln His GlyAsp Asp Pro Tyr Thr Leu Gln 110 115 120 Tyr Gly Gln Cys Gly Asp Arg GlyGln Tyr Ile His Phe Thr Pro 125 130 135 Asn Phe Leu Leu Thr Asp Asn LeuArg Ile Tyr Gly Pro Arg Gly 140 145 150 Arg Val Phe Val His Glu Trp AlaHis Leu Arg Trp Gly Val Phe 155 160 165 Asp Glu Tyr Asn Val Asp Arg SerPro Tyr Ile Ser Arg Lys Asn 170 175 180 Thr Ile Glu Ala Thr Arg Cys SerAla Ser Ile Thr Gly Lys Lys 185 190 195 Val Val His Glu Cys Gln Arg GlySer Cys Val Thr Arg Ala Cys 200 205 210 Arg Arg Asp Ser Lys Thr Arg LeuTyr Glu Pro Lys Cys Thr Phe 215 220 225 Ile Pro Asp Lys Ile Gln Thr AlaGly Ala Ser Ile Met Phe Met 230 235 240 Gln Asn Leu Asn Ser Val Val GluPhe Cys Thr Glu Asn Asn His 245 250 255 Asn Ala Glu Ala Pro Asn Leu GlnAsn Lys Met Cys Asn Arg Arg 260 265 270 Ser Thr Trp Asp Val Ile Lys ThrSer Ala Asp Phe Gln Asn Ala 275 280 285 Pro Pro Met Arg Gly Thr Glu AlaPro Pro Pro Pro Thr Phe Tyr 290 295 300 Leu Leu Lys Ser Arg Arg Arg ValVal Cys Leu Val Leu Asp Lys 305 310 315 Ser Gly Ser Met Asp Lys Glu AspArg Leu Ile Arg Met Asn Gln 320 325 330 Ala Ala Glu Leu Tyr Leu Thr GlnIle Val Glu Lys Glu Ser Met 335 340 345 Val Gly Leu Val Thr Phe Asp SerAla Ala His Ile Gln Asn Tyr 350 355 360 Leu Ile Lys Ile Thr Ser Ser SerAsp Tyr Gln Lys Ile Thr Ala 365 370 375 Asn Leu Pro Gln Gln Ala Ser GlyGly Thr Ser Ile Cys His Gly 380 385 390 Leu Gln Ala Gly Phe Gln Ala IleThr Ser Ser Asp Gln Ser Thr 395 400 405 Ser Gly Ser Glu Ile Val Leu LeuThr Asp Gly Glu Asp Asn Gly 410 415 420 Ile Arg Ser Cys Phe Glu Ala ValSer Arg Ser Gly Ala Ile Ile 425 430 435 His Thr Ile Ala Leu Gly Pro SerArg Ala Arg Glu Leu Glu Thr 440 445 450 Leu Ser Asp Met Thr Gly Gly LeuArg Phe Tyr Ala Asn Lys Asp 455 460 465 Leu Asn Ser Leu Ile Asp Ala PheSer Arg Ile Ser Ser Thr Ser 470 475 480 Gly Ser Val Ser Gln Gln Ala LeuGln Leu Glu Ser Lys Ala Phe 485 490 495 Asp Val Arg Ala Gly Ala Trp IleAsn Gly Thr Val Pro Leu Asp 500 505 510 Ser Thr Val Gly Asn Asp Thr PhePhe Val Ile Thr Trp Met Val 515 520 525 Lys Lys Pro Glu Ile Ile Leu GlnAsp Pro Lys Gly Lys Lys Tyr 530 535 540 Thr Thr Ser Asp Phe Gln Asp AspLys Leu Asn Ile Arg Ser Ala 545 550 555 Arg Leu Gln Ile Pro Gly Thr AlaGlu Thr Gly Thr Trp Thr Tyr 560 565 570 Ser Tyr Thr Gly Thr Lys Ser GlnLeu Ile Thr Met Thr Val Thr 575 580 585 Thr Arg Ala Arg Ser Pro Thr MetGlu Pro Leu Leu Gly Tyr Cys 590 595 600 Tyr Met Ser Gln Ser Thr Ala GlnTyr Pro Ser Arg Met Ile Val 605 610 615 Tyr Ala Arg Val Ser Gln Gly PheLeu Pro Val Leu Gly Ala Asn 620 625 630 Val Thr Ala Leu Ile Glu Ala GluHis Gly His Gln Val Thr Leu 635 640 645 Glu Leu Trp Asp Asn Gly Ala GlyAla Asp Ile Val Lys Asn Asp 650 655 660 Gly Ile Tyr Thr Arg Tyr Phe ThrAsp Tyr His Gly Asn Gly Arg 665 670 675 Tyr Ser Leu Lys Val Arg Val GlnAla Gln Arg Asn Lys Thr Arg 680 685 690 Leu Ser Leu Arg Gln Lys Asn LysSer Leu Tyr Ile Pro Gly Tyr 695 700 705 Val Glu Asn Gly Lys Ile Val LeuAsn Pro Pro Arg Pro Asp Val 710 715 720 Gln Glu Glu Ala Ile Glu Ala ThrVal Glu Asp Phe Asn Arg Val 725 730 735 Thr Ser Gly Gly Ser Phe Thr ValSer Gly Ala Pro Pro Asp Gly 740 745 750 Asp His Ala Arg Val Phe Pro ProSer Lys Val Thr Asp Leu Glu 755 760 765 Ala Glu Phe Ile Gly Asp Tyr IleHis Leu Thr Trp Thr Ala Pro 770 775 780 Gly Lys Val Leu Asp Asn Gly ArgAla His Arg Tyr Ile Ile Arg 785 790 795 Met Ser Gln His Pro Leu Asp LeuGln Glu Asp Phe Asn Asn Ala 800 805 810 Thr Leu Val Asn Ala Ser Ser LeuIle Pro Lys Glu Ala Gly Ser 815 820 825 Lys Glu Ala Phe Lys Phe Lys ProGlu Thr Phe Lys Ile Ala Asn 830 835 840 Gly Ile Gln Leu Tyr Ile Ala IleGln Ala Asp Asn Glu Ala Ser 845 850 855 Leu Thr Ser Glu Val Ser Asn IleAla Gln Ala Val Lys Leu Thr 860 865 870 Ser Leu Glu Asp Ser Ile Ser AlaLeu Gly Asp Asp Ile Ser Ala 875 880 885 Ile Ser Met Thr Ile Trp Gly LeuThr Val Ile Phe Asn Ser Ile 890 895 900 Leu Asn 902 35 18 DNA Artificialsequence Amplification primer 35 gaaccttgcc aggggccg 18 36 22 DNAArtificial sequence Amplification primer 36 ccacgtgctt ctgcgattgc ac 2237 31 DNA Artificial sequence Amplification primer 37 gcggccgcaatggggccatt taagagttct g 31 38 30 DNA Artificial sequence Amplificationprimer 38 gcggccgcag ccctaggcta ttgacagctg 30 39 24 DNA Artificialsequence Amplification primer 39 agaatcaaga tgaacacaga actc 24 40 26 DNAArtificial sequence Amplification primer 40 caaggtattt cacaacttat gacacg26 41 29 DNA Artificial sequence Amplification primer 41 gcggccgctacaacatgacc caaaggagc 29 42 43 DNA Artificial sequence Amplificationprimer 42 gcggccgcga cactttggat atttatttat aataattttg ttc 43 43 19 DNAArtificial sequence Amplification primer 43 cctttatgtt ttgaatgag 19 4422 DNA Artificial sequence Amplification primer 44 caactatgac atctgcctggtc 22 45 25 DNA Artificial sequence Amplification primer 45 cacaaagctaggctaagtca agaac 25 46 903 PRT Unknown Calcium sensitive chloridechannel from bovine tracheal epithelium (Cunningham et al., 1995, J.Biol Chem., 27031016-31026) 46 Met Val Pro Arg Leu Thr Val Ile Leu PheLeu Thr Leu His Leu 5 10 15 Leu Pro Gly Met Lys Ser Ser Met Val Asn LeuIle Asn Asn Gly 20 25 30 Tyr Asp Gly Ile Val Ile Ala Ile Asn Pro Ser ValPro Glu Asp 35 40 45 Glu Lys Leu Ile Gln Asn Ile Lys Glu Met Val Thr GluAla Ser 50 55 60 Thr Tyr Leu Phe His Ala Thr Lys Arg Arg Val Tyr Phe ArgAsn 65 70 75 Val Ser Ile Leu Ile Pro Met Thr Trp Lys Ser Lys Ser Glu Tyr80 85 90 Leu Met Pro Lys Gln Glu Ser Tyr Asp Gln Ala Glu Val Ile Val 95100 105 Ala Asn Pro Tyr Leu Lys His Gly Asp Asp Pro Tyr Thr Leu Gln 110115 120 Tyr Gly Arg Cys Gly Glu Lys Gly Gln Tyr Ile His Phe Thr Pro 125130 135 Asn Phe Leu Leu Thr Asn Asn Leu Pro Ile Tyr Gly Ser Arg Gly 140145 150 Arg Ala Phe Val His Glu Trp Ala His Leu Arg Trp Gly Ile Phe 155160 165 Asp Glu Tyr Asn Gly Asp Gln Pro Phe Tyr Ile Ser Arg Arg Asn 170175 180 Thr Ile Glu Ala Thr Arg Cys Ser Thr His Ile Thr Gly Thr Asn 185190 195 Val Ile Val Lys Cys Gln Gly Gly Ser Cys Ile Thr Arg Pro Cys 200205 210 Arg Arg Asp Ser Gln Thr Gly Leu Tyr Glu Ala Lys Cys Thr Phe 215220 225 Ile Pro Glu Lys Ser Gln Thr Ala Arg Glu Ser Ile Met Phe Met 230235 240 Gln Ser Leu His Ser Val Thr Glu Phe Cys Thr Glu Lys Thr His 245250 255 Asn Val Glu Ala Pro Asn Leu Gln Asn Lys Met Cys Asn Gly Lys 260265 270 Ser Thr Trp Asp Val Ile Met Asn Ser Thr Asp Phe Gln Asn Thr 275280 285 Ser Pro Met Thr Glu Met Asn Pro Pro Thr Gln Pro Thr Phe Ser 290295 300 Leu Leu Lys Ser Lys Gln Arg Val Val Cys Leu Val Leu Asp Lys 305310 315 Ser Gly Ser Met Ser Ser Glu Asp Arg Leu Phe Arg Met Asn Gln 320325 330 Ala Ala Glu Leu Phe Leu Ile Gln Ile Ile Glu Lys Gly Ser Leu 335340 345 Val Gly Met Val Thr Phe Asp Ser Val Ala Glu Ile Arg Asn Asn 350355 360 Leu Thr Lys Ile Thr Asp Asp Asn Val Tyr Glu Asn Ile Thr Ala 365370 375 Asn Leu Pro Gln Glu Ala Asn Gly Gly Thr Ser Ile Cys Arg Gly 380385 390 Leu Lys Ala Gly Phe Gln Ala Ile Ile Gln Ser Gln Gln Ser Thr 395400 405 Ser Gly Ser Glu Ile Ile Leu Leu Thr Asp Gly Glu Asp Asn Glu 410415 420 Ile His Ser Cys Ile Glu Glu Val Lys Gln Ser Gly Val Ile Ile 425430 435 His Thr Val Ala Leu Gly Pro Ser Ala Ala Lys Glu Leu Glu Thr 440445 450 Leu Ser Asp Met Thr Gly Gly His Arg Phe Tyr Ala Asn Lys Asp 455460 465 Ile Asn Gly Leu Thr Asn Ala Phe Ser Arg Ile Ser Ser Arg Ser 470475 480 Gly Ser Ile Thr Gln Gln Thr Ile Gln Leu Glu Ser Lys Ala Leu 485490 495 Ala Ile Thr Glu Lys Lys Trp Val Asn Gly Thr Val Pro Val Asp 500505 510 Ser Thr Ile Gly Asn Asp Thr Phe Phe Val Val Thr Trp Thr Ile 515520 525 Lys Lys Pro Glu Ile Leu Leu Gln Asp Pro Lys Gly Lys Lys Tyr 530535 540 Lys Thr Ser Asp Phe Lys Glu Asp Lys Leu Asn Ile His Ser Ala 545550 555 Arg Leu Arg Ile Pro Gly Ile Ala Glu Thr Gly Thr Trp Thr Tyr 560565 570 Ser Leu Leu Asn Asn His Ala Ser Pro Gln Ile Leu Thr Val Thr 575580 585 Val Thr Thr Arg Ala Arg Ser Pro Thr Thr Pro Pro Val Thr Ala 590595 600 Thr Ala His Met Asn Gln Asn Thr Ala His Tyr Pro Ser Pro Val 605610 615 Ile Val Tyr Ala Gln Val Ser Gln Gly Phe Leu Pro Val Leu Gly 620625 630 Ile Asn Val Thr Ala Ile Ile Glu Thr Glu Asp Gly His Gln Val 635640 645 Thr Leu Glu Leu Trp Asp Asn Gly Ala Gly Ala Asp Ala Thr Lys 650655 660 Asp Asp Gly Val Tyr Ser Arg Tyr Phe Thr Thr Tyr Asp Thr Asn 665670 675 Gly Arg Tyr Ser Val Lys Val His Ala Glu Ala Arg Asn Asn Thr 680685 690 Ala Arg Leu Ser Leu Arg Gln Pro Gln Asn Lys Ala Leu Tyr Ile 695700 705 Pro Gly Tyr Ile Glu Asn Gly Lys Ile Ile Leu Asn Pro Pro Arg 710715 720 Pro Glu Val Lys Asp Asp Leu Ala Lys Ala Glu Ile Glu Asp Phe 725730 735 Ser Arg Leu Thr Ser Gly Gly Ser Phe Thr Val Ser Gly Ala Pro 740745 750 Pro Gly Asn His Pro Ser Val Leu Pro Pro Asn Lys Ile Thr Asp 755760 765 Leu Glu Ala Lys Phe Lys Glu Asp His Ile Gln Leu Ser Trp Thr 770775 780 Ala Pro Ala Asn Val Leu Asp Lys Gly Lys Ala Asn Ser Tyr Ile 785790 795 Ile Arg Ile Ser Lys Ser Phe Leu Asp Leu Gln Lys Asp Phe Asp 800805 810 Asn Ala Thr Leu Val Asn Thr Ser Ser Leu Lys Pro Lys Glu Ala 815820 825 Gly Ser Asp Glu Asn Phe Glu Phe Lys Pro Glu Pro Phe Arg Ile 830835 840 Glu Asn Gly Thr Asn Phe Tyr Ile Ala Val Gln Ala Ile Asn Glu 845850 855 Ala Asn Leu Thr Ser Glu Val Ser Asn Ile Ala Gln Ala Ile Lys 860865 870 Phe Ile Pro Met Pro Glu Asp Ser Val Pro Ala Leu Gly Thr Lys 875880 885 Ile Ser Ala Ile Asn Leu Ala Ile Phe Ala Leu Ala Met Ile Leu 890895 900 Ser Ile Val 903 47 10 PRT Homo sapiens partial sequence of humanc-myc protein 47 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10

We claim:
 1. An isolated nucleic acid molecule comprising a nucleotidesequence encoding a protein having an amino acid sequence of SEQ IDNO:32.
 2. A vector comprising the nucleic acid molecule of claim
 1. 3. Arecombinant vector containing the nucleic acid molecule according toclaim 1, wherein the nucleic acid molecule is operatively linked to oneor more control elements.
 4. A host cell containing the vector of claim3.
 5. A method of providing calcium activated chloride channel activityto a mammalian cell comprising transfecting the mammalian cell with thevector of claim
 3. 6. An isolated and purified polypeptide comprisingthe amino acid sequence of SEQ ID NO:32.
 7. An isolated nucleic acidmolecule comprising a nucleotide sequence of SEQ ID NO:31.